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ObjectiveTo observe the apoptosis induced by paeoniflorin （PF） in non-small cell lung cancer （NSCLC） cells and explore its mechanism. MethodCell counting kit-8 （CCK-8） was used to detect the inhibition rates of H1299， H292 and A549 cells with different concentrations of PF （2.5， 5， 10， 20， 25 µmol·L-1）， and to screen suitable concentrations of PF and experimental cells. The inhibitory effect of PF on lung cancer cells was detected by clone formation assay. The effect of PF on cell apoptosis was detected by flow cytometry with annexin V-FITC/propidium iodide （PI） double staining. With the right concentration of drugs， levels of apoptosis-associated protein B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X protein （Bax）， cleaved Caspase-3 and Caspase-3 were detected by Western blot. At the same time， the molecular expressions of hypoxia inducible factor -1α （HIF-1α） and Hippo signaling pathway were determined. ResultCompared with the blank group， PF significantly inhibited the growth of H1299， H292 and A549 cells of human lung cancer （P<0.01）. PF significantly induced apoptosis in A549 cells （P<0.01）， decreased the Bcl-2/Bax ratio （P<0.01）， and significantly increased the cleaved Caspase-3 expression （P<0.01）. Compared with those in the blank group， the expression levels of HIF-1α， transcriptional coactivator with PDZ-binding motif （TAZ）， large tumor suppressor 1 （LATS1）， Mps one binding 1 （MOB1） and Yes-associated protein （YAP） in A549 cells of the PF treatment group were significantly decreased （P<0.01）， while the expressions of p-LATS1， p-MOB1 and p-YAP were significantly increased （P<0.01）. At the same time， there was no significant effect on the expression levels of phosphorylated mammalian Ste20-like kinase 1 （p-MST1） and MST1， which did not reach a statistical difference. ConclusionAll data demonstrated that PF showed an anti-tumor effect by improving hypoxic conditions and inhibiting the abnormally activated Hippo signaling pathway， thereby inducing and promoting apoptosis in non-small cell lung cancer.
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The vascular endothelial cells (VECs) hypoxia/reoxygenation(H/R) model is a classic cell model that simulates vascular endothelial ischemia/reperfusion (I/R) injury and related diseases. It has the advantages of convenient operation, intuitive image, and good stability, and can accurately reflect pathological changes at the cellular level of diseases. It is widely used in the study of molecular mechanisms of drugs and diseases.There are many similarities in the mechanism and formation between the H/R model and the I/R injury model, but the I/R model is more complex. Therefore, in recent years, many scholars have used the H/R model to simulate the I/R model for experimental research, and believe that the H/R model is also an ideal model for studying I/R. By implementing intervention measures on the established H/R model of VECs, the potential effects of the intervention measures in clinical practice can be verified, which has guiding significance for how to prevent, treat, and how to exacerbate I/R injury in clinical practice. This article introduces the different methods used by scholars in recent years, such as medium deoxygenation and mixed gas culture method, to construct H/R models using VECs cultured in vitro to simulate I/R models. The differences in methods used and the subtle differences between the same methods are also discussed. At the same time, due to the relatively single method of constructing H/R models at present, how to find new, more efficient and affordable methods based on scientific and reasonable experiments has also become a focus of attention.
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Acute kidney injury and acute lung injury/acute respiratory distress syndrome are common in the pediatric intensive care unit.Lung-kidney interaction in critically ill patients is closely related to anoxia, fluid management, and inflammatory response in acute kidney injury and acute lung injury/acute respiratory distress syndrome patients.Strengthening the understanding of lung-kidney interaction can help clinicians to systematically manage critically ill patients.
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Objective:To study the changes of cord blood neutrophil gelatinase-associated lipocalin (NGAL) under different levels of hypoxia at birth and its correlations with hypoxic organ damage (including liver, kidney and heart).Methods:From April to October 2022, all neonates born in our hospital were prospectively enroll in the study. The neonates without perinatal risk factors were assigned into the control group. The neonates with intrauterine distress or hypoxia during labor were assigned into the hypoxia group (no asphyxia at birth) and the asphyxia group (with asphyxia at birth). Cord blood was collected from the umbilical artery and cord blood gas (CBG) and NGAL were measured. Liver enzymes, kidney function and cardiac enzymes as biomarkers for hypoxic organ damage were measured 24~48 h after birth. The correlations of NGAL and the biomarkers were analyzed.Results:A total of 161 neonates were enrolled, including 91 in the control group, 49 in the hypoxia group and 21 in the asphyxia group. NGAL in the asphyxia group was significantly higher than the hypoxia group and the control group [(1.81±0.71) ng/ml vs. (1.22±0.53) ng/ml, (0.88±0.47) ng/ml], NGAL in the hypoxia group significantly higher than the control group ( P<0.05). NGAL was negatively correlated with Apgar score, pH and BE of CBG ( r<-0.3, P<0.05) and positively correlated with lactate, ALT, creatinine ( r>0.3, P<0.05). No significant correlations existed between NGAL and gender, gestational age, birth weight, mother's age, BMI and CK-MB ( P<0.05). ROC curve showed that sensitivity and specificity of NGAL for predicting hypoxic organ damage were 84.3% and 60.3%, respectively, with a cut-off value of 1.07 ng/ml. Conclusions:Cord blood NGAL may increase with the deterioration of hypoxia at birth and may be associated with hypoxic organ damage.
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@#Objective To investigate the protective effect and mechanism of the derivative NIM811 on hypoxia/reoxygenation injury of rat hippocampal neuron (HT22) induced by sodium disulfite (Na2S2O4).Methods The hypoxia/reoxygenation cell model was prepared with mouse HT22 cultured cells.The experiment was divided into normal control group,Na2S2O4 group,Na2S2O4+NIM811 group,and NIM811 group.Cell survival rate was detected by CCK-8,flow cytometry was used to detect apoptosis,mitochondrial membrane potential was detected by JC-1 reagent,calcium ion level in mitochondria was observed by Rhod-2 AM,and reactive oxygen species (ROS) was detected by DCFH-DA method.Results Compared with the Na2S2O4 group,after NIM811 treatment:(1)Cell activity increased by 38% (P<0.01);(2)Apoptosis decreased by 27% (P<0.01);(3)Mitochondrial membrane potential increased (P<0.01);(4)The level of calcium ions in the mitochondria decreased (P<0.01);(5)The level of reactive oxygen species (ROS) decreased (P<0.01).Conclusion NIM811 has a protective effect on the hypoxia/reoxygenation injury of mouse hippocampal neurons caused by Na2S2O4.The mechanism may be related to the maintenance of mitochondrial homeostasis and inhibition of cell apoptosis.NIM811 has therapeutic potential for future clinical treatment of ischemic stroke.
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Hypoxia is a common phenomenon of solid tumor, which is closely related to the malignant proliferation, tumor progression, radiotherapy and chemotherapy resistance, treatment failure, and poor prognosis. At present, many researches focus on the application of medical imaging and nuclear medicine methods in detecting the hypoxic areas of tumors. This article focuses on the detection of hypoxia microenvironment and the application of PET tracers in tumor hypoxia imaging.
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Objective To explore the risk stratification of pulse oxygen saturation (SpO2) in patients with emergency hypoxemia patients, and to provide evidence for the identification of critical illness. Methods Self-designed clinical data registration form for patients with emergency hypoxemia , and prospective collection of 344 hypoxemia patients in the emergency department of Peking Union Medical College Hospital from March to April in 2018, including baseline data (name, gender, age, ID number, date, registration time), hospitalization method, past history, patient complaint and diagnosis, triage level, SpO2, whether to enter the rescue room, etc. Results All of 344 emergency hypoxemia patients, there were 163 cases (21.2% ) of ambulances, and 107 cases (31.1% ) of patients requiring immediate rescue. There were 54 cases (25.7%) and 53 cases (39.6%) in need of immediate rescue in day shift (8:00-20:00) and night shift (20:00-8:00 next day), with 9:00-10:00, 14:00-15:00, 20:00-24:00 in the majority. There was a statistical difference in the way of hospitalization, triage, and SpO(2 25.780, t=4.722, all P<0.05). SpO2 risk stratification was 0.905 in patients without pulmonary disease, and SpO2 risk stratification in patients with lung disease was 0.765. Conclusions Patients with hypoxemia account for a certain proportion in the emergency department and are in critical condition. The degree of critical condition of patients can be evaluated based on whether they have lung diseases, and the risk stratification of patients can be accurately determined with the help of SpO2, so as to further guide the hierarchical treatment measures for patients with emergency hypoxemia and rationally optimize the χ2=29.537, allocation of emergency resources.
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Objective To study the effects of hypoxia on the expression of inflammatory factor high mobility group box-l(HMGB1) in the pulmonary arteriolae of neonatal SD rats.Method A total of 80 neonatal SD rats were randomly assigned into control group and hypoxia-induced persistent pulmonary hypertension of the newborn model (PPHN) group.The PPHN group was subdivided into 2 h,8 h,24 h,and 3 d post-PPHN subgroups according to the time of sacrifice.PPHN model was established on postnatal day 4 when rat pups in PPHN group were kept in low-oxygen box (10％ O2 and 90％ N2) for consecutively 7 days.Multi-channel physiological transducer RM-6280 was used recording the mean pulmonary artery pressure (mPAP) at the root to pulmonary artery of rat pups.ELISA method was used examining the serum level of HMGB1 of rat pups in each group.The pathology of the lung tissue was studied using optical microscope after HE staining,and MIAS-2000 medical image analysis software was used to calculate the ratio of the middle membrane thickness to the outer diameter of the pulmonary arteriolae wall (MT％).Protein level of HMGB1 in the lung was examined using Western Blot.Result The lung pathology in PPHN rats showed thickening of the middle membrane of the pulmonary arteriolae wall and stenosis of the pulmonary arteriolae.MT％ of control group and PPHN group were 5.3％ (3.7％,7.6％) and 7.1％ (4.6％,9.2％),respectively,without significant differences (P＞0.05).At 2 h,8 h,24 h,3 d post-PPHN timepoints,the serum levels of HMGB1 in PPHN group were (13.2±3.1),(15.4±3.6),(17.1±3.5),and (15.8±3.6) ng/ml,respectively,without intra-subgroup differences (F=2.134,P＞0.05),but significant differences existed when compared with control group at each timepoint (P＜0.01).Western Blot showed that HMGB1 protein expression in the lungs were significantly elevated soon after PPHN,peaked at 8～24 h,and reduced but still significantly elevated at 3 d after PPHN comparing with normal control.Significant differences existed at 2 h,8 h,and 24 h timepoints (P＜0.01,respectively).The HMGB1 protein of PPHN group declined significantly at 3 d timepoint without significant differences comparing with the control group (P＞0.05).Conclusion HMGB1 is closely related with the pathogenesis of PPHN,indicating the inflammatory response plays an important role in the mechanisms of PPHN.HMGB1 may be an indicator for the assessment of hypoxia-induced PPHN.
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Objective To investigate the pathways by which hypoxia aggravates the neurotoxic effects of amyloid-beta protein (Aβ) on neurons.Methods Human neuroblastoma cells (SH-SY5Y cells) were cultured in vitro,and were divided into four groups:the control group,Aβ intervention group,hypoxia group,Aβ intervention plus hypoxia group.Quantitative real time polymerase chain reaction(qRT-PCR) was adopted to detect the mRNA expression levels of p21-activated kinase 1 (PAK1),LIM kinase 1 protein (LIMK1)and cofilin.Western blot was used to measure the protein levels of PAK1,LIMK1,phosphate-LIMK1 (P-LIMK1),cofilin and phosphate-cofilin (P-cofilin).Results After Aβ treatment,the activity of SH-SY5Y cells was decreased.Compared with the control group,the protein levels of PAK1,LIMK1,P-LIMK1,P-cofilin,and the mRNA expression levels of PAK1 and LIMK1 were decreased(all P＜0.05),but the protein and mRNA expression of cofilin had no significant changes after 24 h of treatment with 10μmol/L Aβ.Compared with the Aβ intervention group,the protein levels of PAK 1,LIMK1,P-LIMK 1 and P-cofilin were decreased (all P ＜ 0.05),and the mRNA expression levels of PAK1 and LIMK1 were decreased(both P＜0.05),but the protein and mRNA expression of cofilin had no significant changes after 24 h of treatment of SH-SY5Y cells with 10 μmol/L Aβ plus 2％ oxygen.Conclusions Aβ may reduce P-LIMK1 expression by inhibiting the activity of PAK1,thereby reducing the P-cofilin,increasing the formation of dephosphorylated cofilin,leading to neural cells damage,and hypoxia aggravates the neurotoxicity of Aβ through this pathway.
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Objective@#To explore the risk stratification of pulse oxygen saturation (SpO2) in patients with emergency hypoxemia patients, and to provide evidence for the identification of critical illness.@*Methods@#Self-designed clinical data registration form for patients with emergency hypoxemia, and prospective collection of 344 hypoxemia patients in the emergency department of Peking Union Medical College Hospital from March to April in 2018, including baseline data (name, gender, age, ID number, date, registration time), hospitalization method, past history, patient complaint and diagnosis, triage level, SpO2, whether to enter the rescue room, etc.@*Results@#All of 344 emergency hypoxemia patients, there were 163 cases (21.2%) of ambulances, and 107 cases (31.1%) of patients requiring immediate rescue. There were 54 cases (25.7%) and 53 cases (39.6%) in need of immediate rescue in day shift (8:00-20:00) and night shift (20:00-8:00 next day), with 9:00-10:00, 14:00-15:00, 20:00-24:00 in the majority. There was a statistical difference in the way of hospitalization, triage, and SpO2 (χ2=29.537, 25.780, t=4.722, all P<0.05) . SpO2 risk stratification was 0.905 in patients without pulmonary disease, and SpO2 risk stratification in patients with lung disease was 0.765.@*Conclusions@#Patients with hypoxemia account for a certain proportion in the emergency department and are in critical condition. The degree of critical condition of patients can be evaluated based on whether they have lung diseases, and the risk stratification of patients can be accurately determined with the help of SpO2, so as to further guide the hierarchical treatment measures for patients with emergency hypoxemia and rationally optimize the allocation of emergency resources.
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Objective@#To investigate the protective effects and mechanism of keratinocyte growth factor (KGF) combined with hypoxia inducible factor-1α (HIF-1α) on intestinal crypt epithelial cells (IEC-6) of rats with hypoxia stress.@*Methods@#(1) The routinely cultured IEC-6 of rats were collected and divided into normoxia blank group, normoxia KGF group, normoxia HIF-1α group, and normoxia combine group, according to the random number table, and then the previous mediums were respectively replaced with dulbecco′s modified eagle medium (DMEM), medium with 0.5 ng/mL KGF, medium with 10.0 ng/mL HIF-1α, and medium with 0.5 ng/mL KGF and 30.0 ng/mL HIF-1α. And the cells were cultured in cell incubator with oxygen volume fraction of 21% for 24 hours. (2) Another batch of routinely cultured IEC-6 were collected and divided into normoxia control group, hypoxia control group, hypoxia KGF group, hypoxia HIF-1α group, and hypoxia combine group, according to the random number table. The previous mediums were replaced with DMEM, DMEM, medium with 0.5 ng/mL KGF, medium with 10.0 ng/mL HIF-1α, and medium with 0.5 ng/mL KGF and 30.0 ng/mL HIF-1α respectively. And then, the cells in normoxia control group were cultured routinely for 24 hours, and cells in the other 4 groups were cultured in cells incubator of 3 gases, with oxygen volume fraction of 5% for 24 hours. Cells cultured in normoxic and hypoxic incubators were collected, with 3 samples in each group, and morphological changes of cells were observed with optical microscope. Cells cultured in normoxic and hypoxic incubators were collected, with 3 samples in each group, and survival rates of cells were detected by cell count kit 8. Cells in normoxia control group and cells cultured in hypoxic incubator were collected, with 3 samples in each group. The cell cycle changes and apoptosis rates were detected by flow cytometer, the content of adenosine triphosphate (ATP) was detected by ultraviolet spectrophotometer, and protein expression of p53 was detected by Western blotting. Data were processed with one-way analysis of variance and least significant difference test.@*Results@#(1) After being cultured for 24 h, cells cultured in normoxic incubator grew well with oval or round shapes and clear cytoplasm, and cells cultured in hypoxic incubator showed irregular shapes such as fusiform or starlike shape, with black particle in cytoplasm. (2) After being cultured for 24 h, cell survival rates of normoxia blank group, normoxia KGF group, normoxia HIF-1α group, and normoxia combine group were (107.4±8.7)%, (109.8±2.9)%, (115.8±7.4)%, and (112.8±10.6)% respectively. There was no significantly statistical difference in general comparison of cell survival rates among the above groups (F=0.685, P=0.586). After being cultured for 24 h, cell survival rates of hypoxia control group, hypoxia KGF group, hypoxia HIF-1α group, and hypoxia combine group were (35.1±4.6)%, (52.9±6.8)%, (56.2±3.1)%, and (71.2±9.6)% respectively, which were significantly lower than (106.3±12.3)% of normoxia control group (P<0.001). Survival rates of cells in hypoxia KGF group, hypoxia HIF-1α group, and hypoxia combine group were significantly higher than the rate of cells in hypoxia control group (P=0.023, 0.009, <0.001). Survival rate of cells in hypoxia combine group was significantly higher than the rates of cells in hypoxia KGF group and hypoxia HIF-1α group (P=0.017, 0.045). (3) After being cultured for 24 h, percentage of cells in G1 phase in hypoxia control group was significantly higher than that of cells in normoxia control group (P=0.030), percentages of cells in S phase in hypoxia control group, hypoxia KGF group, and hypoxia HIF-1α group were obviously lower than the percentage of cells in normoxia control group (P=0.020, 0.031, 0.026), and percentages of cells in different phases in other groups were close to those of cells in normoxia control group (P=0.516, 0.107, 0.052, 0.985, 0.637, 0.465, 0.314, 0.591). After being cultured for 24 h, percentages of cells in G1 phase in hypoxia control group, hypoxia KGF group, and hypoxia HIF-1α group were obviously higher than the percentage of cells in hypoxia combine group (P=0.001, 0.030, 0.014), and percentages of cells in S phase in the above 3 groups were obviously lower than the percentage of cells in hypoxia combine group (P=0.001, 0.012, 0.010). (4) After being cultured for 24 h, compared with that of cells in normoxia control group, apoptosis rate of cells in hypoxia control group obviously increased (P=0.018), and apoptosis rate of cells in hypoxia combine group obviously decreased (P=0.008). After being cultured for 24 h, compared with that of cells in hypoxia control group, apoptosis rates of cells in hypoxia KGF group and hypoxia combine group obviously decreased (P=0.004, 0.001). Apoptosis rate of cells in hypoxia combine group was obviously lower than those of cells in hypoxia KGF group and hypoxia HIF-1α group (P=0.032, 0.002). (5) After being cultured for 24 h, compared with that of cells in normoxia control group, the content of ATP of cells in hypoxia combine group changed unobviously (P=0.209), and content of ATP of cells in the other groups obviously decreased (P= <0.001, 0.001, 0.002). Content of ATP of cells in hypoxia HIF-1α group and hypoxia combine group was obviously higher than that of cells in hypoxia control group (P=0.044, 0.001). Content of ATP of cells in hypoxia combine group was obviously higher than that of cells in hypoxia KGF group and hypoxia HIF-1α group (P=0.011, 0.020). (6) After being cultured for 24 h, protein expressions of p53 of cells in hypoxia control group, hypoxia KGF group, and hypoxia HIF-1α group were obviously higher than that of cells in normoxia control group (P<0.001), and protein expression of p53 of cells in hypoxia combine group was obviously lower than those of cells in hypoxia control group, hypoxia KGF group, and hypoxia HIF-1α group (P=0.001, 0.001, 0.002).@*Conclusions@#KGF combined with HIF-1α have significant protective effects on IEC-6 of rats with hypoxia stress, and can improve its survival in hypoxic environment by inhibiting cell cycle arrest, reducing the level of apoptosis, and increasing level of energy metabolism.
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Objective@#To investigate the role of hexokinase Ⅱ in the changes of autophagic flow in cardiomyocytes of mice with ischemia-hypoxia in vitro.@*Methods@#The hearts of totally six male and female C57BL/6 mice aged from 1 to 2 days were isolated to culture primary cardiomyocytes which were used for the following experiments. (1) The cells were divided into 6 groups according to the random number table (the same grouping method below), i. e., normal control 3, 6, and 9 h groups and ischemia-hypoxia 3, 6, and 9 h groups, with 4 wells in each group. After being regularly cultured for 48 h with Dulbecco′s modified Eagle medium/nutrient mixture F12 (DMEM/F12) medium (the same regular culture condition below), the cells in normal control 3, 6, and 9 h groups were cultured with replaced fresh DMEM/F12 medium for 3, 6, and 9 h, respectively, and the cells in ischemia-hypoxia 3, 6, and 9 h groups were cultured with replaced sugar-free serum-free medium in the low-oxygen incubator with a volume fraction of 1% oxygen and a volume fraction of 5% carbon dioxide at 37 ℃ (the same hypoxic culture condition below) for 3, 6, and 9 h, respectively. Cell viability was measured by the cell counting kit 8 (CCK-8) method. (2) The cells were grouped and treated the same as those in experiment (1), with 1 well in each group. Western blotting was used to detect the protein expressions of microtubule-associated protein 1 light chain 3 Ⅰ (LC3Ⅰ), LC3Ⅱ, p62, and hexokinase Ⅱ. (3) The cells were divided into normal control group, simple ischemia-hypoxia 9 h group, and ischemia-hypoxia 9 h+ 2-deoxyglucose (2-DG) group, with 4 wells in each group. After a regular culture for 48 h, the cells in normal control group were cultured with replaced fresh DMEM/F12 medium for 9 h; the cells in simple ischemia-hypoxia 9 h group were replaced with sugar-free serum-free medium, and the cells in ischemia-hypoxia 9 h+ 2-DG group were replaced with sugar-free serum-free medium in which 2-DG was dissolved in a concentration of 10 mmol/L (20 μmol), and then they were cultured with hypoxia for 9 h. Cell viability was measured by CCK-8 method. (4) The cells were grouped and treated the same as those in experiment (3), with 1 well in each group. Western blotting was used to detect the protein expressions of LC3Ⅰ, LC3Ⅱ, and p62. (5) The cells were grouped and treated the same as those in experiment (3), with 2 wells in each group. Transmission electron microscope was used to observe autophagosomes/autolysosomes in cardiomyocytes. (6) The cells were divided into normal control group, simple ischemia-hypoxia 9 h group, ischemia-hypoxia 9 h+ hexosinase Ⅱ small interfering RNA1 (HK-ⅡsiRNA1) group, and ischemia-hypoxia 9 h+ HK-ⅡsiRNA2 group, with 4 wells in each group. The cells in normal control group and simple ischemia-hypoxia 9 h group were regularly cultured for 48 h, and the cells in ischemia-hypoxia 9 h+ HK-ⅡsiRNA1 group and ischemia-hypoxia 9 h+ HK-ⅡsiRNA2 group were respectively transfected with 200 nmol/L HK-ⅡsiRNA1 and HK-ⅡsiRNA2 and then also cultured for 48 h. The cells in normal control group were cultured with replaced fresh DMEM/F12 medium for 9 h, and the cells in simple ischemia-hypoxia 9 h group, ischemia-hypoxia 9 h+ HK-ⅡsiRNA1 group, and ischemia-hypoxia 9 h+ HK-ⅡsiRNA2 group were cultured with replaced sugar-free serum-free medium and hypoxia for 9 h. Cell viability was measured by CCK-8 method. (7) The cells were grouped and treated the same as those in experiment (6), with 1 well in each group. Western blotting was used to detect the protein expressions of LC3Ⅰ, LC3Ⅱ, p62, and hexokinase Ⅱ. Except for experiment (5), each experiment was repeated 3 times. Data were processed with one-way analysis of variance and lest significant difference t test, and Bonferroni correction.@*Results@#(1) The viabilities of cardiomyocytes in ischemia-hypoxia 3, 6, and 9 h groups were 0.450±0.022, 0.385±0.010, and 0.335±0.015, respectively, which were significantly lower than 0.662±0.026, 0.656±0.028, and 0.661±0.021 of the corresponding normal control 3, 6, and 9 h groups, respectively (t=6.21, 9.12, 12.48, P<0.01). (2) Compared with those of corresponding normal control 3, 6, and 9 h groups, the LC3Ⅱ/Ⅰ ratio and protein expressions of p62 and hexokinase Ⅱ in cardiomyocytes of ischemia-hypoxia 3, 6, and 9 h groups were significantly increased (t3 h=16.15, 10.99, 5.30, t6 h=6.79, 10.42, 9.42, t9 h=15.76, 16.51, 7.20, P<0.05 or P<0.01). (3) The viability of cardiomyocytes in simple ischemia-hypoxia 9 h group was 0.353±0.022, which was significantly lower than 0.673±0.027 of normal control group (t=9.29, P<0.01). The viability of cardiomyocytes in ischemia-hypoxia 9 h+ 2-DG group was 0.472±0.025, which was significantly higher than that of simple ischemia-hypoxia 9 h group (t=3.60, P<0.05). (4) Compared with those of normal control group, the LC3Ⅱ/Ⅰ ratio and protein expression of p62 in cardiomyocytes of simple ischemia-hypoxia 9 h group were significantly increased (t=9.45, 8.40, P<0.01). Compared with those of simple ischemia-hypoxia 9 h group, the LC3Ⅱ/Ⅰratio and protein expression of p62 in cardiomyocytes of ischemia-hypoxia 9 h+ 2-DG group were significantly decreased (t=4.39, 4.74, P<0.05). (5) In cardiomyocytes of normal control group, only single autophagosome/autolysosome with bilayer membrane structure was observed. Compared with that of normal control group, the number of autophagosome/autolysosome with bilayer membrane structure in cardiomyocytes of simple ischemia-hypoxia 9 h group was increased significantly. Compared with that of simple ischemia-hypoxia 9 h group, the number of autophagosome/autolysosome with bilayer membrane structure in cardiomyocytes of ischemia-hypoxia 9 h+ 2-DG group was significantly decreased. (6) The viability of cardiomyocytes in simple ischemia-hypoxia 9 h group was 0.358±0.023, which was significantly lower than 0.673±0.026 in normal control group (t=9.12, P<0.01). The viabilities of cardiomyocytes in ischemia-hypoxia 9 h+ HK-ⅡsiRNA1 group and ischemia-hypoxia 9 h+ HK-ⅡsiRNA2 group were 0.487±0.027 and 0.493±0.022, respectively, which were significantly higher than the viability in simple ischemia-hypoxia 9 h group (t=3.63, 4.28, P<0.05). (7) Compared with those of normal control group, the LC3Ⅱ/Ⅰratio and protein expressions of p62 and hexokinase Ⅱ in cardiomyocytes of simple ischemia-hypoxia 9 h group were significantly increased (t=6.08, 6.31, 4.83, P<0.05 or P<0.01). Compared with those of simple ischemia-hypoxia 9 h group, the LC3Ⅱ/Ⅰ ratio and protein expressions of p62 and hexokinase Ⅱ in cardiomyocytes of ischemia-hypoxia 9 h+ HK-ⅡsiRNA1 group and ischemia-hypoxia 9 h+ HK-ⅡsiRNA2 group were significantly decreased (t=5.10, 7.76, 15.33, 4.17, 8.42, 12.11, P<0.05 or P<0.01).@*Conclusions@#Ischemia-hypoxia upregulates the expression level of hexokinase Ⅱ protein in mouse cardiomyocytes cultured in vitro, which decreases the viability of cardiomyocytes by impairing autophagic flow. To inhibit the activity of hexokinase Ⅱ or its expression can alleviate the ischemia-hypoxia damage of cardiomyocytes.
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Objective@#To explore the effects of transient receptor potential vanilloid 1 (TRPV1) on autophagy in early hypoxic mouse cardiomyocytes and the mechanism in vitro.@*Methods@#The hearts of 120 C57BL/6 mice aged 1-2 days, no matter male or female, were isolated, and then primary cardiomyocytes were cultured and used for the following experiments, the random number table was used for grouping. (1) The cells were divided into normoxia group and hypoxia 3, 6, and 9 h groups, with one well in each group. The cells in normoxia group were routinely cultured (the same below), the cells in hypoxia 3, 6, and 9 h groups were treated with fetal bovine serum-free and glucose-free Dulbecco′ s modified Eagle medium under low oxygen condition in a volume fraction of 1% oxygen, 5% carbon dioxide, and 94% nitrogen for 3, 6, and 9 h, respectively. The protein expressions of microtubule-associated protein 1 light chain 3 (LC3), Beclin-1, TRPV1 were determined with Western botting. (2) The cells were divided into normoxia group and hypoxia group, with two coverslips in each group. The cells in hypoxia group were treated with hypoxia for 6 h as above. The positive expression of TRPV1 was detected by immunofluorescence assay. (3) The cells were divided into 4 groups, with one well in each group. The cells in simple hypoxia group were treated with hypoxia for 6 h as above, and the cells in hypoxia+ 0.1 μmol/L capsaicin group, hypoxia+ 1.0 μmol/L capsaicin group, and hypoxia+ 10.0 μmol/L capsaicin group were respectively treated with 0.1, 1.0, 10.0 μmol/L capsaicin for 30 min before hypoxia for 6 h. The protein expressions of LC3, Beclin-1, and TRPV1 were detected by Western blotting. (4) The cells were divided into 5 groups, with 5 wells in each group. The cells in hypoxia group were treated with hypoxia for 6 h as above, the cells in hypoxia+ chloroquine group, hypoxia+ capsaicin group, and hypoxia+ capsaicin+ chloroquine group were treated with hypoxia for 6 h after being cultured with 50 μmol/L chloroquine, 10.0 μmol/L capsaicin, and 50 μmol/L chloroquine+ 10.0 μmol/L capsaicin for 30 min, respectively. Viability of cells was detected by cell counting kit 8 assay. (5) The cells were divided into simple hypoxia group and hypoxia+ 10.0 μmol/L capsaicin group, with one well in each group. The cells in hypoxia group were treated with hypoxia for 6 h as above, the cells in hypoxia+ 10.0 μmol/L capsaicin group were treated with 10.0 μmol/L capsaicin for 30 minutes and then with hypoxia for 6 h. The protein expressions of lysosomal associated membrane protein 1 (LAMP-1) and LAMP-2 were detected by Western blotting. Each experiment was repeated for 3 or 5 times. Data were processed with one-way analysis of variance, least significant difference t test, and Bonferroni correction.@*Results@#(1) Compared with those of normoxia group, the protein expressions of LC3, Beclin-1, and TRPV1 were significantly increased in cardiomyocytes of hypoxia 3, 6, and 9 h groups (t3 h=4.891, 5.890, 4.928; t6 h=9.790, 6.750, 10.590; t9 h=6.948, 6.764, 5.049, P<0.05 or P<0.01), which of hypoxia 6 h group were the highest (1.08±0.05, 1.12±0.10, 0.953±0.071, respectively). (2) The density of TRPV1 in cell membrane and inside the cardiomyocytes in hypoxia group was significantly increased with lump-like distribution, and the expression of TRPV1 was higher than that in normoxia group. (3) Compared with those of simple hypoxia group, the protein expression of Beclin-1 in cardiomyocytes of hypoxia+ 0.1 μmol/L capsaicin group was increased (t=10.488, P<0.01), while the protein expressions of LC3 and TRPV1 were increased without statistically significant differences (t=4.372, 3.026, P>0.05); the protein expressions of LC3, TRPV1, and Beclin-1 in cardiomyocytes of hypoxia+ 1.0 μmol/L capsaicin group and hypoxia+ 10.0 μmol/L capsaicin group were significantly increased (t=15.505, 5.773, 13.430; 20.915, 8.054, 16.384; P<0.05 or P<0.01), which of hypoxia+ 10.0 μmol/L capsaicin group were the highest (2.33±0.09, 1.34±0.07, 1.246±0.053, respectively). (4) Compared with 0.585±0.045 in normoxia group, the cardiomyocyte viability in hypoxia group was significantly decreased (0.471±0.037, t=4.365, P<0.05). Compared with that in hypoxia group, the cardiomyocyte viability in hypoxia+ chloroquine group was further decreased (0.350±0.023, t=6.216, P<0.01), while 0.564±0.047 in hypoxia+ capsaicin group was significantly increased (t=3.489, P<0.05). Compared with that in hypoxia+ chloroquine group, the cardiomyocyte viability in hypoxia+ capsaicin+ chloroquine group did not significantly change (0.364±0.050, t=0.545, P>0.05). (5) Compared with 0.99±0.04 and 0.54±0.04 in simple hypoxia group, the protein expressions of LAMP-1 and LAMP-2 in hypoxia+ 10.0 μmol/L capsaicin group were significantly increased (1.49±0.06, 0.81±0.05, t=12.550, 7.442, P<0.01).@*Conclusions@#TRPV1 can further promote the expression of autophagy-related proteins in hypoxic cardiomyocytes through autophagy-lysosomal pathway, enhance autophagy activity, and improve autophagic flow for alleviating early hypoxic cardiomyocyte injury.
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Objective@#To understand the mechanism of chemotherapy resistance in nasopharyngeal carcinoma under hypoxic conditions through the perspective of protein SUMOylation modification.@*Methods@#Cobalt chloride (CoCl2) was used to establish the hypoxic model of human nasopharyngeal carcinoma CNE1 cells. Then, the cell cycle was detected by flow cytometry, and the expression level of small ubiquitin-related modifier(SUMO) and cyclin-dependent kinase 6 (CDK6) proteins were detected by western blotting. MTT assay was used to determine the median lethal dose (IC50) of cancer cells against cisplatin, and enzyme-linked immunosorbent assay (ELISA) was used to determine lactate dehydrogenase (LDH) level.@*Results@#The cell cycle of CNE1 induced by hypoxia was arrested in G0/G1 phase.The results of Western blot showed that the protein expression level of CDK6 in CNE1 cells was lower than that in the control group (0.83±0.25 vs. 0.43±0.21, t=14.67, P=0.003). The protein level of conjugated SUMO1 was significantly lower than that in the control group (2.69±0.48 vs. 1.38±0.31, t=17.22, P=0.001), while the level of free SUMO1 protein was significantly higher than that in the control group (2.01±0.43 vs. 2.60±0.59, t=15.45, P=0.002).The LC50 of CNE1 cells in the control group was significantly lower than that in the hypoxic group (29.44 μg/ml vs. 97.72 μg/ml, t=12.79, P=0.001). After CNE1 cells received 50 μg/ml cisplatin for 48 h, the LDH content in the supernatant of the control group was significantly higher than that in the hypoxic group ((541.49±64.59) ng/ml vs. (234.67±41.03) ng/ml, t=11.94, P=0.007)). The apoptosis rate of CNE1 cells in the control group was significantly higher than that in the hypoxic group ((76.64±5.37)% vs. (32.84±4.77) ng/ml, t=8.49, P=0.003)).@*Conclusion@#Hypoxia can dissociate the covalent modification of CDK6 and SUMO1, inhibit cell cycle and increase the chemotherapy resistance of nasopharyngeal carcinoma.
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Objective@#To investigate blood pressure and vascular remodeling of OSAS by establishing the chronic-intermittent hypoxia model in rat.@*Methods@#Experiments were performed on 35 adult male Sprague-Dawley rats. Animals were randomly divided into four groups: unhandled control group (with 5 rats in it), CIH group at 9/6/3 weeks (with 10 ratsin each group). Rats in CIH group went through 8-hour intermittent hypoxia everyday, and those in control group were raising normally. After 9-week experiment, blood pressure was measured. The changes of the following indexes were observed: pathological changes of aorta and the middle aorta thickness (HE staining), the collagen of aorta wall (Masson staining). The experimental data were analyzed by SPSS 24.0 statistical software. The variance was analyzed by one-way analysis of variance, and the irregularity was selected using the calibration t test.@*Results@#The systolic and diastolic blood pressures of the CIH9, 6, and 3 weeks groups and the control group were: (127±13) and (79±9), (124±11) and (81±7), (101±11) and (75±9), (91±10) and (65±9) mmHg (1 mmHg=0.133 kPa). The systolic blood pressure and diastolic blood pressure of the rats in the week of CIH 9 and 6 weeks were significantly higher than the control group (F=14.64, P=0.000; F=6.81, P=0.000). There was no significant difference in the mean blood pressure between the three groups of CIH and the control group. Membrane thickness in CIH9, 6 and 3 weeks and control group were: (20±2), (19±2), (14±2), (13±3) μm. Compared with the control group, the aortic pathology and thickness of the middle layer of the CIH9 and 6 weeks group were significantly thicker (F=20.24, P=0.000), but there was no significant difference between the CIH3 week group and the control group; the collagen deposition was unchanged compared with the control group.@*Conclusion@#Intermittent hypoxia for 6 weeks or more in rats resulted in the increasement of blood pressure, morphological changes of aorta and vascular remodeling in thickened media.
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[Abstract] China has a vast territory and often needs workers in a special environment. Working and training in a special environment (such as high temperature, plateau anoxia, cold, long voyage, and so on) will put forward higher requirements for the body, and the body will also have adaptive changes. However, once the compensation range of the body is exceeded, there will be a series of discomfort and even pathological changes. A small number of literatures have described the major changes of the whole body under various special conditions, but there is still a lack of related summary of the metabolic changes, compensatory changes and pathological changes of kidney under various extreme environments. Therefore, the compensatory modification of the kidney and the possible causes of injury in various special environments have been summarized and analyzed in present paper, and combined with the related pathophysiological mechanism, the related protective measures of kidney have also been introduced.
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Stroke is a leading cause of death worldwide. Up to one thousand potential drugs or interventions have been developed to treat stroke, out of which ~160 have gone on to clinical trials. However, none of them has been successful. New insights into the molecular and cellular mechanisms of ischemia-induced injury are needed for discovering new therapeutic targets. Recently, Drosophila has been used to uncover new hypoxia-related genes. In this study, we describe an efficient and reliable assay with a sophisticated apparatus for studying the effects of oxygen deprivation on flies. Using this assay, wild-type flies were exposed to an anoxic environment for varying lengths of time, then the cumulative death rate and mobility recovery were systematically analyzed. We found that anoxia for over one hour caused lethality. The cumulative death rate on day 5 after anoxia was linearly and positively correlated with the duration of anoxia, and reached 50% when the duration was 2.5 h-3 h. We also found that the mobility recovery in normoxia was slow, as the climbing ability remained largely unchanged 4 h-6 h after 2.5-h of anoxia. We suggest that 2.5 h-3 h of anoxia and 4 h-6 h of recovery before mobility analysis are appropriate for future use of the anoxia assay.
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Animales , Conducta Animal , Modelos Animales de Enfermedad , Drosophila melanogaster , HipoxiaRESUMEN
Objective To evaluate the efficacy of intermittent positive pressure ventilation (IPPV,1 ml/kg) of the operated lungs in preventing hypoxemia during one-lung ventilation (OLV) in elderly patients undergoing radical resection of esophageal cancer.Methods Sixty American Society of Anesthsiologists physical status Ⅰ or Ⅱ patients of both sexes,aged 65-75 yr,with body mass index of 18.5-24.0 kg/m2,were divided into 2 groups (n =30 each) using a random number table method:convention group and IPPV group.In convention group,ventilator settings were adjusted with the tidal volume of 6-8 ml/kg,respiratory rate of 15 breaths/min,inspiratory/expiratory ratio of 1 ∶ 2,and fraction of inspired oxygen 70％ during OLV.In IPPV group,ventilator settings were adjusted with the tidal volume of 1 ml/kg,respiratory rate of 15 breaths/min,and fraction of inspired oxygen 70％ on the operated side during OLV,and the ventilator settings on the other side were consistent with those previously described in convention group.Before anesthesia induction (T0),at 10 min of two-lung ventilation (T1),at 15,30 and 45 min of OLV (T2,4) and at 10 min after the end of OLV (T5),blood samples were collected from the radial artery for blood gas analysis.The partial pressure of arterial oxygen (PaO2) and alveolar to arterial partial pressure of oxygen were recorded.Respiratory index (RI) and oxygenation index (OI) were calculated.The occurrence of PaO2 ＜ 60 mmHg,RI＞ 1.0 and OI＜ 200 mmHg was recorded during OLV.Results Compared with convention group,the RI was significantly decreased at T4,the PaO2 and OI were increased,and the incidence of PaO2＜60 mmHg,RI＞ 1.0 and OI ＜ 200 mmHg was decreased in IPPV group (P＜ 0.05).Conclusion IPPV (1 ml/kg) of the operated lungs can prevent the occurrence of hypoxemia during OLV in elderly patients undergoing radical resection of esophageal cancer.
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Aim To study the effect of puerarin( Pue) against myocardial ischemia/reperfusion injury and in-volved mitochondrial mechanism. Methods Anoxia/reoxygenation( A/R) injury model was established in H9c2 cell. Recombinant plasmid pFLAG-VDAC1 was constructed. Cells were randomly divided into 4 groups, normal control group ( Control) , A/R group, puerarin group ( Pue + A/R ) , and pFLAG-VDAC1-Pue group. Real-time PCR was used to investigate the expression of VDAC1 at mRNA level, and the expres-sion of protein level was detected by Western blot. LDH and CK activities were measured by automatic bi-ochemical analyzer. Mitochondrial membrane potential ( Δψm) and cell apoptosis were observed by flow cy-tometry method. Mitochondrial swelling test was used to detect the opening of mitochondria permeability tran- sition pore ( mPTP) . Results Compared with control group, the expression of VDAC1 and mRNA was up-regulated in A/R group, LDH and CK activity were el-evated, and then mPTP opened, Δψm collapsed, cell apoptosis was significantly increased. Puerarin pre-treatment can lower the expression of VDAC1, main-tain Δψm, prevent the opening of mPTP, and reduce apoptosis. However, the protective effect of Puerarin could be cancelled by transfection of pFLAG-VDAC1. Conclusions The cardioprotection of Puerarin against A/R injury is closely related to down-regulation of VDAC1 and prevention of mPTP opening.
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Objective To prospectively investigate the effects of high-flow nasal oxygen on hypoxemic patients after surgery.Methods A total of 108 postoperation hypoxemic patients (150 mmHg ≤ PaO2/FiO2 ＜300 mmHg, PaCO2 ≤50 mmHg) in Beijing Hospital Surgical Intensive Critical Unit was in cluded and assigned randomly to two groups from June 2016 to April 2017.Fifty four patients (study group) who received high-flow nasal oxygen therapy were compared with 54 patients (control group) who received noninvasive ventilation therapy.The data of reintubation and mortality in 28 days after extubation were collected and analyzed.Results No significant differences were found for reintubation rate (11.1％ vs 13.0％,P =0.767) and mortality (5.6％ vs 7.4％, P =0.696) in 28 days after extubation between two groups.In subgroup analysis, no significant differences were found for different hypoxima level (250 mmHg ≤ PaO2/FiO2 ＜ 300 mmHg,200 mmHg ≤ PaO2/FiO2 ＜ 250 mmHg and 150 mmHg≤ PaO2/FiO2 ＜ 200 mmHg) between two groups, for reintubation rate (0 vs 3.7％,P =0.296;20％ vs 14.3％,P =0.684;30％ vs 30.8％,P =0.968, respectively) and mortality (0 vs 3.7％,P =0.296;6.7％ vs 7.1％,P =0.960;20％ vs 15.4％,P =0.772, respectively).Face skin breakdown were significantly more common in control group (1 1.1％ vs 0,P =0.012).Conclusions High-flow nasal oxygen therapy was not inferior to noninvasive ventilation for mild and moderate hypoxemic patients after surgery.High-flow nasal oxygen therapy is safe and effective for these patients.