Peroxiredoxin 1 alleviates oxygen-glucose deprivation/ reoxygenation injury in N2a cells via suppressing the JNK/caspase-3 pathway.

Objectives: Cerebral ischemia/reperfusion (I/R) injury inevitably aggravates the initial cerebral tissue damage following a stroke. Peroxiredoxin 1 (Prdx1) is a representative protein of the endogenous antioxidant enzyme family that regulates several reactive oxygen species (ROS)-dependent signaling pathways, whereas the JNK/caspase-3 proapoptotic pathway has a prominent role during cerebral I/R injury. This study aimed to examine the potential mechanism of Prdx1 in Neuro 2A (N2a) cells following oxygen-glucose deprivation and reoxygenation (OGD/R) injury. Materials and Methods: N2a cells were exposed to OGD/R to simulate cerebral I/R injury. Prdx1 siRNA transfection and the JNK inhibitor (SP600125) were used to interfere with their relative expressions. CCK-8 assay, flow cytometry, and lactate dehydrogenase (LDH) assay were employed to determine the viability and apoptosis of N2a cells. The intracellular ROS content was assessed using ROS Assay Kit. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analyses were conducted to detect the expression levels of Prdx1, JNK, phosphorylated JNK (p-JNK), and cleaved caspase-3. Results: Firstly, Prdx1, p-JNK, and cleaved caspase-3 expression were significantly induced in OGD/R-exposed N2a cells. Secondly, the knockdown of Prdx1 inhibited cell viability and increased apoptosis rate, expression of p-JNK, and cleaved caspase-3 expression. Thirdly, SP600125 inhibited the JNK/caspase-3 signaling pathway and mitigated cell injury following OGD/R. Finally, SP600125 partially reversed Prdx1 down-regulation-mediated cleaved caspase-3 activation and OGD/R damage in N2a cells. Conclusion: Prdx1 alleviates the injury to N2a cells induced by OGD/R via suppressing JNK/caspase-3 pathway, showing promise as a potential therapeutic for cerebral I/R injury.

Effect and mechanism of miRNA-92a regulating SHH pathway on promoting vascular regeneration after myocardial I/R injury / 中华老年心脑血管病杂志

Objective To explore the effect and mechanism of miR-92a regulating sonic hedgehog(SHH)pathway on promoting vascular regeneration after myocardial ischemia-reperfusion(I/R)injury.Methods Primary cardiomyocytes were isolated from newborn SD rats(1 to 3 days old),and then cultured to establish a cellular model of hypoxia/reoxygenation injury.The cardiomyo-cytes were divided into cardiomyocyte normoxia group and cardiomyocyte I/R group.After miR-92a mimic and inhibitor were respectively transfected into primary cardiomyocytes to overex-press or lower its expression,the cells were then grouped into control,I/R,miR-92a mimic and in-hibitor groups.CCK-8 assay was used to determine cell viability,flow cytometry was employed to detect cell apoptosis,ELISA and QT-PCR were applied to detect the expression of VEGF,b-FGF and Ang-1,and Western blotting was performed to measure the expression of SHH signaling pathway related proteins.Results The expression level of miR-92a was significantly higher in the cardiomyocytes from the ischemia/reperfusion(I/R)group than the normoxia group(3.89±0.29 vs 1.53±0.19,P＜0.01).Statistical differences were observed among the control group,miR-92a inhibitor group,I/R group,and miR-92a mimic group in the protein levels of SHH(0.57±0.13 vs 0.51±0.11 vs 0.24±0.03 vs 0.14±0.02,P＜0.01),of Smoothened(SMO,0.53±0.12 vs 0.49± 0.10 vs 0.14±0.04 vs 0.09±0.01,P＜0.01),of glioma-associated oncogene homolog 1(Gli-1,0.56±0.14 vs 0.50±0.13 vs 0.15±0.03 vs 0.08±0.01,P＜0.01),and of glioma-associated onco-gene homolog 2(Gli-2,0.58±0.11 vs 0.49±0.12 vs 0.18±0.02 vs 0.11±0.03,P＜0.01).Conclu-sion MiR-92a is abnormally highly expressed in cardiomyocytes after I/R injury,and inhibition of miR-92a can activate SHH signaling pathway to promote the expression of angiogenesis factors effectively.

Role of autophagy in morphine preconditioning-induced reduction of OGD/R injury in primary cortical neurons of mice and the relationship with JNK / 中华麻醉学杂志

Objective:To evaluate the role of autophagy in morphine preconditioning-induced reduction of oxygen-glucose deprivation and restoration (OGD/R) injury in primary cortical neurons of mice and the relationship with c-Jun N-terminal kinase (JNK).Methods:Primary cortical neurons extracted from C57BL/6 neonatal mice within 24 h after birth were divided into 9 groups ( n=24 each) using a random number table method: control group (C group), OGD/R group, morphine preconditioning group (M group), autophagy inhibitor 3-methyladenine (3-MA) group (3-MA group), 3-MA+ morphine preconditioning group (3-MA+ M group), autophagy inhibitor chloroquine group (Ch group), chloroquine + morphine preconditioning group (Ch+ M group), JNK inhibitor SP600125 group (SP group) and SP600125 + morphine preconditioning group (SP+ M group). Morphine preconditioning: morphine was added at a final concentration of 3 μmol/L before OGD/R, and the cells were incubated for 2 h in OGD/R group. In 3-MA, Ch and SP groups, 3-MA 5 mmol/L, chloroquine 50 μmol/L and SP600125 25 μmol/L were added, respectively, and the cells were incubated for 150 min. In 3-MA+ M, Ch+ M and SP+ M groups, 3-MA 5 mmol/L, chloroquine 50 μmol/L and SP600125 25 μmol/L were added, respectively, at 30 min before morphine preconditioning. Then the cells were subjected to oxygen-glucose deprivation for 1 h followed by restoration of oxygen-glucose supply for 24 h. CCK-8 assay was used to detect the neuronal viability. The expression of JNK, phosphorylated JNK (p-JNK), microtubule-associated protein 1 light chain 3 (LC3), p62, Beclin1, caspase-3, and cleaved-caspase-3 was determined by Western blot. The autophagosomes and autolysosomes were counted using LC3-double fluorescent adenovirus transfection, and the neuronal apoptosis rate was determined by TUNEL staining. Results:Compared with C group, the neuronal viability was significantly decreased, the expression of Beclin1 was up-regulated, the expression of p62 was down-regulated, and the LC3Ⅱ/LC3Ⅰ ratio, p-JNK/JNK ratio, the number of autophagosomes and autolysosomes, cleaved-caspase-3/caspase-3 ratio and neuronal apoptosis rate were increased in OGD/R group ( P<0.001). Compared with OGD/R group, the neuronal viability, p-JNK/JNK ratio, LC3Ⅱ/LC3Ⅰ ratio and the number of autophagosomes and autolysosomes were significantly increased, the expression of Beclin1 was up-regulated, and the expression of p62 was down-regulated in M group, the LC3Ⅱ/LC3Ⅰ ratio was significantly decreased, and the expression of p62 was down-regulated in 3-MA group, the LC3Ⅱ/LC3Ⅰ ratio was significantly increased, and the expression of p62 was up-regulated in Ch group ( P<0.001), and no significant change was found in the parameters mentioned above in SP600125 group ( P>0.05). Compared with M group, the neuronal viability was significantly decreased, the LC3Ⅱ/LC3Ⅰ ratio was decreased, and the expression of p62 was up-regulated in M+ 3-MA group, the neuronal viability was significantly decreased, the LC3Ⅱ/LC3Ⅰ ratio was increased, and the expression of p62 was up-regulated in M+ Ch group, and the neuronal viability, LC3Ⅱ/LC3Ⅰ ratio and p-JNK/JNK ratio were significantly decreased, the expression of p62 was up-regulated, the number of autophagosomes and autolysosomes was decreased, the expression of Beclin1 was down-regulated, and the cleaved-caspase-3/caspase-3 ratio and neuronal apoptosis rate were increased in M+ SP group ( P<0.001). Conclusions:Morphine preconditioning can attenuate OGD/R injury by activating JNK, enhancing autophagy and inhibiting apoptosis in primary cortical neurons of mice.

Effect of Notch1 signaling on cellular proliferation and apoptosis in human laryngeal carcinoma.

BACKGROUND: The occurrence and development of malignancies include excessive proliferation and apoptosis resistance in tumor cells. This study aimed to identify the effects of Notch1 signaling on proliferation and apoptosis of laryngeal cancer cells in a hypoxic microenvironment. METHODS: Notch1 and Ki-67 expression in laryngeal squamous cell carcinoma (LSCC) tissues was detected by immunohistochemistry. The apoptotic index (AI) of LSCC was evaluated by the TUNEL method. Small interfering RNA (siRNA) was used to inhibit Notch1 expression in laryngeal cancer cells. Real-time PCR was used to measure Notch1, Hes1, and Hey1 mRNA expression, and Western blotting was used to measure Notch1 and Notch1 intracellular domain (N1ICD) protein expression. Annexin V-FITC/propidium iodide staining and Cell Counting Kit-8 assays were used to measure cell apoptosis and proliferation, respectively. RESULTS: Notch1 expression was significantly related to the proliferation index (PI) and AI in LSCC tissues. Hypoxia could induce proliferation and inhibit apoptosis in cancer cells. Notch1 expression and Notch1 signaling activity could be upregulated by hypoxia. Suppressing Notch1 signaling activity in hypoxic cells could decrease proliferation and increase apoptosis. CONCLUSIONS: Our study has demonstrated that hypoxia may promote proliferation and inhibit apoptosis of laryngeal cancer cells. Notch1 signaling may play a pivotal role in regulating the proliferation and apoptosis resistance of laryngeal cancer cells under hypoxic conditions.

Unique hypoxia-tolerant subpopulations of adipose-derived stem cells: ITGB3+ cells.

BACKGROUND: We found by accident that stem cells could still be isolated from adipose tissue stored for 14 days in sealed tubes, which was distinct from previous protocols. The morphology of these hypoxia-tolerant stem cells also differs from that of conventional adipose-derived stem cells (ADSCs). In this study, we aim to define the newly found subsets. MATERIALS AND METHODS: Stem cells were isolated from adipose tissue that was aspirated immediately or stored for 14 days. The stem cells were then harvested for flowcytometric analysis and differentiation potentials. The expression of hypoxia-inducible factor 1 alpha (HIF-1α) was assayed to confirm the hypoxia-tolerant ability. RNA sequencing (RNA-seq) was performed to find the common signatures of the hypoxia-tolerant cells. The result of bioinformatics was tested by quantitative real-time reverse transcription-polymerase chain reaction (qPCR) and western blotting. RESULTS: Certain subsets of ADSCs can be isolated from adipose tissue stored for 14 days. These survived cells were positive for CD90, CD105, and CD73 and showed multilineage differentiation potentials. The hypoxic condition was evidenced by up-regulation of HIF-1α for 2.0-fold changes (p < 0.05). The hypoxia-tolerant stem cells were distinct from multilineage-differentiating stress-enduring (Muse) cells, previously found stress-enduring stromal cells. RNA-seq suggested that integrin beta 3 (ITGB3) was highly expressed in hypoxia-tolerant subpopulations. The result was further confirmed at transcription and translation levels by qPCR and western blotting (mRNA: 2.9 ± 0.4, p < 0.05; protein: 1.5 ± 0.2, p < 0.05; respectively). The conventional ADSCs are positive for ITGB3, which implies that ITGB3+ cells are subpopulations of heterogeneous ADSCs. CONCLUSIONS: Our study reveals the ITGB3+ subsets with potent hypoxia tolerance, which has significant implications for improving fat retention rates and curing obesity-related diseases.

Notch1 signaling modulates hypoxia-induced multidrug resistance in human laryngeal cancer cells.

BACKGROUND: Laryngeal carcinoma is one of the common malignant tumors of the head and neck. Multidrug resistance (MDR) remains a critical problem in the chemotherapy of patients with laryngeal cancer. This study aims to clarify the role and mechanisms of Notch1 signaling in MDR induced by hypoxia in laryngeal cancer cells. METHODS AND RESULTS: Laryngeal carcinoma cells were cultured under normoxia or hypoxia. Notch1 expression was inhibited by small interfering RNA (siRNA). The mRNA expression of Notch1, Hes1, Hey1, MDR1 and survivin was analyzed by real-time PCR. The protein expression of Notch1, the Notch1 intracellular domain (N1ICD), MDR1/P-gp and survivin was analyzed by Western blotting. Current research has shown that hypoxia can upregulate Notch1 expression and Notch1 signaling activity. Furthermore, suppression of Notch1 expression effectively downregulated Notch1 signaling activity and the expression of the MDR and survivin genes in laryngeal cancer cells under hypoxic conditions (P < 0.05). The Cell Counting Kit-8 (CCK-8) assay results confirmed that the sensitivity of hypoxic laryngeal cancer cells to a variety of drugs could be upregulated by suppressing Notch1 expression (P < 0.05). Additionally, flow cytometry (FCM) showed that suppression of Notch1 expression significantly increased drug-induced apoptosis and intracellular rhodamine 123 (Rh123) accumulation in hypoxic laryngeal carcinoma cells (P < 0.05). CONCLUSIONS: Notch1 signalling could be regarded as a pivotal regulator of hypoxia-induced MDR in laryngeal cancer cells through the regulation of survivin-mediated apoptosis resistance and MDR1/P-gp-mediated drug transport.

Hypoxia-preconditioning of human adipose-derived stem cells enhances cellular proliferation and angiogenesis: A systematic review.

BACKGROUND: Human adipose-derived stem cells (hADSCs) have gained attention lately because of their ease of harvesting and ability to be substantially multiplied in laboratory cultures. Stem cells are usually cultured under atmospheric conditions; however, preconditioning stem cells under hypoxic conditions seems beneficial. AIM: This systematic review aims to investigate the effect of hypoxia preconditioning and its impact on the proliferation and angiogenic capacity of the hADSCs. METHODS: We performed a systematic review by searching PubMed, Scopus, Embase, and Google Scholar databases from all years through March 22, 2021, following Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Medical Subject Headings terms "adipose-derived stem cell," "Hypoxia," "cell proliferation," and "angiogenesis" guided our search. Only articles written in English using experimental models comparing a preconditioned group against a control group of hADSCs with data on proliferation and angiogenic capacity were included. RESULTS: Our search yielded a total of 321 articles. 11 articles met our inclusion criteria and were ultimately included in this review. Two studies induced hypoxia using hypoxia-inducible factor-1 alpha stabilizing agents, while nine reached hypoxia by changing oxygen tension conditions around the cells. Four articles conducted in-vivo studies to correlate their in-vitro findings, which proved to be consistent. Although 1 article indicated cell proliferation inhibition with hypoxia preconditioning, the remaining 10 found enhanced proliferation in preconditioned groups compared to controls. All articles showed an enhanced angiogenic capacity of hADSCs after hypoxia preconditioning. CONCLUSION: In this review, we found evidence to support hypoxia preconditioning of hADSCs before implantation. Benefits include enhanced cell proliferation with a faster population doubling rate and increased secretion of multiple angiogenic growth factors, enhancing angiogenesis capacity. RELEVANCE FOR PATIENTS: Although regenerative therapy is a promising field of study and treatment in medicine, much is still unknown. The potential for angiogenic therapeutics with stem cells is high, but more so, if we discover ways to enhance their natural angiogenic properties. Procedures and pathologies alike require the assistance of angiogenic treatments to improve outcome, such is the case with skin grafts, muscle flaps, skin flaps, or myocardial infarction to mention a few. Enhanced angiogenic properties of stem cells may pave the way for better outcomes and results for patients.

Therapeutic approach for digestive system cancers and potential implications of exercise under hypoxia condition: what little is known? a narrative review.

BACKGROUND: Cancer, like other chronic pathologies, is associated with the presence of hypoxic regions due to the uncontrolled cell growth. Under this pathological hypoxic condition, various molecular signaling pathways are activated to ensure cell survival, such as those that govern angiogenesis, erythropoiesis, among others. These molecular processes are very similar to the physiological response caused by exposure to altitude (natural hypobaric systemic hypoxia), the use of artificial hypoxia devices (systemic normobaric simulated hypoxia) or the delivery of vascular occlusion to the extremities (also called local hypoxia by the blood flow restriction technique). "Tumor hypoxia" has gained further clinical importance due to its crucial role in both tumor progression and resistance to treatment. However, the ability to manipulate this pathway through physical exercise and systemic hypoxia-mediated signaling pathways could offer an important range of therapeutic opportunities that should be further investigated. METHODS: This review is focused on the potential implications of systemic hypoxia combined with exercise in digestive system neoplasms prognosis. Articles included in the review were retrieved by searching among the three main scientific databases: PubMed, Scopus, and Embase. FINDINGS: The findings of this review suggest that exercise performed under systemic hypoxic conditions could have a positive impact in prognosis and quality of life of the population with digestive system cancers. CONCLUSIONS: Further studies are needed to consider this paradigm as a new potential intervention in digestive oncological population.

Augmented secretion of IL-1α from mouse oral squamous cell carcinoma (OSCC) vcells caused by serum deprivation and hypoxia promotes immune suppressive activity of mesenchymal stromal cells.

OBJECTIVES: We have previously reported that mouse oral squamous carcinoma (OSCC), Sq-1979-1, produces interleukin (IL)-1α, which specifically enhances the immunosuppressive activity of co-cultured mesenchymal stromal 10T1/2 cells. This study assessed the conditions promoting the production of IL-1α in Sq-1979-1 cells, which could further enhance the immunosuppressive function of 10T1/2 cells, and evaluated its expression in OSCC tissues. METHODS: The expression of IL-1α was examined by RT-PCR, western blotting, and enzyme-linked immune sorbent assay (ELISA). The interferon (IFN)- γ-producing capability of anti-CD3 antibody-stimulated mouse spleen cells co-cultured with 10T1/2 cells and conditioned medium (CM) from Sq-1979-1 cells was examined by ELISA. The function of IL-1α was examined using an anti-IL1α antibody. Immunohistochemical analysis of the OSCC tissues was performed. RESULTS: The production of IL-1α from Sq-1979-1 cells was synergistically enhanced in lower serum (0.5% or 1.0% FBS) at the transcriptional level, and under hypoxia (1.0% oxygen) at the release level compared to that in the control medium supplemented with 10% FBS under normoxia. The IFN-γ-producing capability of stimulated spleen cells co-cultured with 10T1/2 cells was significantly reduced in the CMs prepared with the lower serum or under hypoxia. These functions of CMs were completely abolished by the anti-IL-1α antibody. The expression of IL-1α in OSCC tissues was prominent in the midst of a carcinomatous cellular lesion or a nearby necrotic lesion, where a supply deficiency could occur. CONCLUSION: s: IL-1α production by Sq-1979-1 cells was synergistically augmented under low serum and hypoxic conditions, which could promote the immunosuppressive activity of mesenchymal cells.

Hyperbaric oxygen therapy to prevent central airway stenosis after lung transplantation.

BACKGROUND: Central airway stenosis (CAS) is a severe airway complication after lung transplantation associated with bronchial ischemia and necrosis. We sought to determine whether hyperbaric oxygen therapy (HBOT), an established treatment for tissue ischemia, attenuates post-transplant bronchial injury. METHODS: We performed a randomized, controlled trial comparing usual care with HBOT (2 atm absolute for 2 hours × 20 sessions) in subjects with extensive airway necrosis 4 weeks after transplantation. Endobronchial biopsies were collected at 4, 7, and 10 weeks after transplantation for a quantitative polymerase chain reaction. Coprimary outcomes were incidence of airway stenting and acute cellular rejection (ACR) at 1 year. RESULTS: The trial was stopped after enrolling 20 subjects (n = 10 per group) after a pre-planned interim analysis showed no difference between usual care and HBOT groups in stenting (both 40%), ACR (70% and 40%, respectively), or CAS (40% and 60%, respectively). Time to first stent placement (median [interquartile range]) was significantly shorter in the HBOT group (150 [73-150] vs 186 [167-206] days, p < 0.05). HIF gene expression was significantly increased in donor tissues at 4, 7, and 10 weeks after transplantation but was not altered by HBOT. Subjects who developed CAS or required stenting had significantly higher HMOX1 and VEGFA expression at 4 weeks (both p < 0.05). Subjects who developed ACR had significant FLT1, TIE2, and KDR expression at 4 weeks (all p < 0.05). CONCLUSIONS: Incidence of CAS is high after severe, established airway necrosis after transplantation. HBOT does not reduce CAS severity or stenting. Elevated HMOX1 and VEGFA expressions appear to associate with airway complications.

[Effects of B-cell lymphoma-2/adenovirus E1B 19 000 interacting protein 3 on the migration and motility of human dermal microvascular endothelial cells under hypoxia and the mechanism].

Objective: To explore the effects of B-cell lymphoma-2/adenovirus E1B 19 000 interacting protein 3 (BNIP3) on the migration and motility of human dermal microvascular endothelial cells (HDMECs) under hypoxia and the mechanism. Methods: The experimental research method was applied. (1) HDMECs were divided into normoxia group received routine culture and hypoxia 6, 12, 24 h groups treated under hypoxia with oxygen volume fraction of 2% for corresponding time according to the random number table (the same grouping method below). Western blotting was used to detect the protein expressions of BNIP3 and microtubule-associated protein 1 light chain 3Ⅱ (LC3Ⅱ) in HDMECs. (2) HDMECs were divided into normoxia+ unloaded group, normoxia+ BNIP3 knockdown group, hypoxia+ unloaded group, and hypoxia+ BNIP3 knockdown group which were transfected with unloaded virus or BNIP3 knockdown virus and were subjected to normoxic or hypoxic treatment. The BNIP3 protein expression was detected by Western blotting and immunofluorescence staining. The scratch area at 24 h post scratching was detected by scratch test, and the healing rate of scratch was calculated. The curve distance of cell movement was measured with the living cell workstation, and the speed of movement was calculated within 3 hours. (3) HDMECs were grouped and treated as experiment (2). Western blotting and immunofluorescence staining were performed to detect the protein expression of LC3Ⅱ. The number of sample was 3 in the above-mentioned experiments. Data were statistically analyzed with one-way analysis of variance and least significant difference test. Results: (1) Compared with those of normoxia group, the protein expressions of BNIP3 and LC3Ⅱ of cells in hypoxia 6, 12, 24 h groups were significantly increased (P<0.01). (2) After 6 hours of culture, compared with that of hypoxia+ unloaded group, the BNIP3 protein expressions of cells in normoxia+ unloaded group and hypoxia+ BNIP3 knockdown group were significantly decreased (P<0.05 or P<0.01). The red fluorescence denoting BNIP3 protein expression of cells in normoxia+ unloaded group and normoxia+ BNIP3 knockdown group was weak, the red fluorescence of cells in hypoxia+ unloaded group was strong, and the red fluorescence of cells in hypoxia+ BNIP3 knockdown group was significantly decreased compared with that in hypoxia+ unloaded group. After scratching for 24 hours, the scratch of cells in hypoxia+ unloaded group basically healed, while the remaining scratch area in the other three groups were large. The healing rates of scratch of cells in normoxia+ unloaded group, normoxia+ BNIP3 knockdown group, hypoxia+ unloaded group, and hypoxia+ BNIP3 knockdown group were (61±4)%, (58±4)%, (88±4)%, and (57±4)%, respectively. The healing rate of scratch of cells in hypoxia+ unloaded group was significantly higher than that in normoxia+ unloaded group (P<0.01) and hypoxia+ BNIP3 knockdown group (P<0.05). Within 3 hours of observation, the range of cell movement in hypoxia+ unloaded group was significantly larger than that in normoxia+ unloaded group, the range of cell movement in hypoxia+ BNIP3 knockdown group was significantly smaller than that in hypoxia+ unloaded group, and the curve movement velocity of cells in hypoxia+ unloaded group was significantly higher than that in normoxia+ unloaded group and hypoxia+ BNIP3 knockdown group (P<0.01). (3) After 6 hours of culture, compared with hypoxia+ unloaded group, the LC3Ⅱ protein expressions of cells in hypoxia+ unloaded group and hypoxia+ BNIP3 knockdown group were decreased significantly (P<0.05 or P<0.01). After 6 hours of culture, the red fluorescence denoting LC3 protein expressions of cells was weak in normoxia+ unloaded group and normoxia+ BNIP3 knockdown group, the red fluorescence of cells was significantly enhanced in hypoxia+ unloaded group, and the red fluorescence of cells was significantly inhibited in hypoxia+ BNIP3 knockdown group. Conclusions: BNIP3 can promote the migration and motility of HDMECs under hypoxia, and autophagy may be involved in the regulation migration of HDMECs by BNIP3.

Effect of TBK1 overexpression on hypoxia-reoxygenation injury in isolated mouse cardiomyocytes subjected to high glucose: relationship with mitochondrial autophagy / 中华麻醉学杂志

Objective:To evaluate the effect of TBK1 overexpression on hypoxia-reoxygenation (H/R) injury in isolated mouse cardiomyocytes subjected to high glucose and the relationship with mitochondrial autophagy.Methods:Normally cultured log-phase HL-1 mouse cardiomyocytes were inoculated in a 6-well plate at a density of 1×10 6 cells/ml and were divided into 4 groups ( n=10 each) using a random number table method: control group (group C), high glucose group (group HG), high glucose and H/R group (group HG+ H/R), and TBK1 overexpression group (group TBK1). The cells were incubated in culture medium with 1% fetal bovine serum and 1% double antibody for 24 h when the cell density reached 50%.When the cell density reached 80%, pcDNA3.1 (+ ) was used as a vector to achieve TBK1 overexpression.The cells were cultured with high glucose medium (33 mmol/L) for 24 h, exposed to 94% N 2+ 5% CO 2+ 1% O 2 for 24 h in an incubator at 37℃ followed by 12 h reoxygenation in an incubator containing 5% CO 2 at 37°C to establish the model of H/R injury to cardiomyocytes subjected to high glucose.After reoxygenation, CCK-8 assay was used to detect cell viability, the activity of lactic dehydrogenase (LDH) in supernatant was detected using LDH kit, mitochondrial contents were determined using Mito-Tracter green fluorescent probe, and the expression of TBK1 and mitophagy-related proteins PINK1, Parkin, LC3B and P62 was detected by Western blot. Results:Compared with group C, the cell viability was significantly decreased, the activity of LDH in supernatant was increased, mitochondrial contents were decreased, the expression of TBK1, PINK1, Parkin and LC3B was down-regulated, and the expression of P62 was up-regulated in HG group and HG+ H/R group ( P<0.05). Compared with group HG, the cell viability was significantly decreased, the activity of LDH in supernatant was increased, mitochondrial contents were decreased, the expression of TBK1, PINK1, Parkin and LC3B was down-regulated, and the expression of P62 was up-regulated in group HG+ H/R ( P<0.05). Compared with group HG+ H/R, the the cell viability was significantly increased, the activity of LDH in supernatant was decreased, mitochondrial contents were increased, the expression of TBK1, PINK1, Parkin and LC3B was up-regulated, and the expression of P62 was down-regulated in group TBK1 ( P<0.05). Conclusion:The mechanism by which TBK1 overexpression reduces the H/R injury is related to restoring mitophagy in isolated mouse cardiomyocytes subjected to high glucose.

Increased ratio of P[v-a]CO2 to C[a-v]O2 without global hypoxia: the case of metformin-induced lactic acidosis.

The ratio of venoarterial CO2 tension to arteriovenous O2 content difference (P[v-a]CO2/C[a-v]O2) increases when lactic acidosis is due to inadequate oxygen supply (hypoxia); we aimed to verify whether it also increases when lactic acidosis develops because of mitochondrial dysfunction (dysoxia) with constant oxygen delivery. Twelve anaesthetised, mechanically ventilated pigs were intoxicated with IV metformin (4.0 to 6.4 g over 2.5 to 4.0 h). Saline and norepinephrine were used to preserve oxygen delivery. Lactate and P[v-a]CO2/C[a-v]O2 were measured every one or two hours (arterial and mixed venous blood). During metformin intoxication, lactate increased from 0.8 (0.6-0.9) to 8.5 (5.0-10.9) mmol/l (p < 0.001), even if oxygen delivery remained constant (from 352 ± 78 to 343 ± 97 ml/min, p = 0.098). P[v-a]CO2/C[a-v]O2 increased from 1.6 (1.2-1.8) to 2.3 (1.9-3.2) mmHg/ml/dl (p = 0.004). The intraclass correlation coefficient between lactate and P[v-a]CO2/C[a-v]O2 was 0.72 (p < 0.001). We conclude that P[v-a]CO2/C[a-v]O2 increases when lactic acidosis is due to dysoxia. Therefore, a high P[v-a]CO2/C[a-v]O2 may not discriminate hypoxia from dysoxia as the cause of lactic acidosis.

Type I collagen facilitates safe and reliable expansion of human dental pulp stem cells in xenogeneic serum-free culture.

BACKGROUND: Human dental pulp stem cells (DPSCs) are a readily accessible and promising cell source for regenerative medicine. We recently reported that a xenogeneic serum-free culture medium (XFM) is preferable to fetal bovine serum-containing culture medium for ex vivo expansion of DPSCs; however, we observed that, upon reaching overconfluence, XFM cells developed a multilayered structure and frequently underwent apoptotic death, resulting in reduced cell yield. Therefore, we focused on optimization of the XFM culture system to avoid the undesirable death of DPSCs. METHODS: We selected type I collagen (COL) as the optimal coating substrate for the cultureware and compared DPSCs cultured on COL in XFM (COL-XFM cells) to the conventional XFM cultures (XFM cells). RESULTS: Our results demonstrated that COL coating facilitated significantly higher rates of cell isolation and growth; upon reaching overconfluence, cell survival and sustained proliferative potential resulted in two-fold yield compared to the XFM cells. Surprisingly, after subculturing the overconfluent COL-XFM cultures, the cells retained stem cell behavior including stable cell growth, multidifferentiation potential, stem cell phenotype, and chromosomal stability, which was achieved through HIF-1α-dependent production and uniform distribution of collagen type I and its interactions with integrins α2ß1 and α11ß1 at overconfluency. In contrast, cells undergoing apoptotic death within overconfluent XFM cultures had disorganized mitochondria with membrane depolarization. CONCLUSION: The use of COL as a coating substrate promises safe and reliable handling of DPSCs in XFM culture, allowing translational stem cell medicine to achieve stable isolation, expansion, and banking of donor-derived stem cells.

Role of hypoxia-related proteins in adenoid cystic carcinoma invasion.

BACKGROUND: Among cancers affecting the oral cavity, adenoid cystic carcinoma (ACC) is a relatively common malignant neoplasm. It has high rates of metastasis and recurrence and is associated with significant morbidity. During the progression of ACC, the oxygen concentration is reduced in specific areas of the tumour microenvironment, leading to intratumoural hypoxia. The expression of NOTCH1, a disintegrin and metalloproteinase 12 (ADAM-12), hypoxia-inducible factor 1 alpha (HIF-1α), and heparin-binding epidermal growth factor (HB-EGF) under hypoxic conditions has been implicated in invadopodia formation, tumour invasiveness, and metastasis. The aim of this study was to analyse the expression of these proteins to elucidate the mechanisms underlying ACC invasiveness. METHODS: Fifteen ACC samples and 10 normal-looking salivary gland (SG) samples were used to investigate the expression of these proteins by immunohistochemistry. Primary antibodies against NOTCH1, ADAM-12, HIF-1α, and HB-EGF were used. RESULTS: The immunoexpression of all proteins was higher in ACC samples than in SG samples (p < 0.05). CONCLUSIONS: There was increased expression of proteins associated with hypoxia and tumour invasiveness in ACC samples, which indicates a possible role of these proteins in the biological behaviour of this tumour.

Relación entre el índice gradiente dióxido de carbono/gradiente arteriovenoso de oxígeno y lactato en el choque séptico por neumonía / Relationship between carbon dioxide gradient/arteriovenous oxygen gradient index and lactate in septic shock secondary to pneumonia

RESUMEN Objetivo Determinar la relación del índice gradiente de dióxido de carbono/gradiente arteriovenoso de oxígeno y lactato en pacientes con neumonía complicada y choque séptico en áreas críticas del Hospital III EsSalud Chimbote durante el periodo 2018 y 2019. Materiales y métodos Estudio de tipo cuantitativo, observacional, correlacional y longitudinal de tendencias. La unidad de análisis está constituida por los pacientes con neumonía complicada por choque séptico que presentan lecturas de índice de gradiente dióxido de carbono / gradiente arteriovenoso de oxígeno y lecturas de lactato. Resultados Se incluyeron 90 pacientes con neumonía complicada por choque séptico. El presente trabajo de investigación demuestra una fuerte relación entre el índice de gradiente de gases y el lactato sérico: al inicio, 3 horas, 6 horas y a las 12 horas, de iniciado y monitorizado estos pacientes. Conclusiones El índice del gradiente de dióxido de carbono/gradiente concentración arterio venoso de oxígeno > 1.4, se relaciona con el lactato sérico, en pacientes con shock séptico por neumonía.

ABSTRACT Objective To determine the relationship between carbon dioxide gradient/arteriovenous oxygen gradient index and lactate in patients with pneumonia complicated by septic shock in areas delivering care for critically ill or injured patients of the Hospital III EsSalud Chimbote between 2018 and 2019. Materials and methods A quantitative, observational, correlational, longitudinal trend study. The study population consisted of patients with pneumonia complicated by septic shock who had carbon dioxide gradient/arteriovenous oxygen gradient index and lactate readings. Results Ninety (90) patients with pneumonia complicated by septic shock participated in the research. The present study showed a robust relationship between gas gradient index and serum lactate when monitoring patients at baseline, 3 hours, 6 hours and 12 hours. Conclusions There is a relationship between carbon dioxide gradient/arteriovenous oxygen gradient index (> 1.4) and serum lactate levels in patients with septic shock secondary to pneumonia.

Lower limit of adequate oxygen delivery for the maintenance of aerobic metabolism during cardiopulmonary bypass in neonates.

BACKGROUND: The objective of cardiopulmonary bypass (CPB) is to maintain an adequate balance between oxygen delivery (Do2) and consumption. The critical Do2 is that at which consumption becomes supply dependent. This study aimed to identify the critical Do2 in neonates, who have higher metabolic rates than adults. METHODS: In a retrospective cohort of neonates, Do2 was calculated from CPB parameters recorded during aortic cross-clamping. High lactate concentration measured after aortic unclamping (lactOFF) was used to identify anaerobic metabolism. Data were analysed using mixed linear and proportional odds regression models. The relationship between Do2 and temperature was analysed in a subgroup of patients with lactOFF <2.5 mM, thought to have had balanced oxygen delivery and consumption. The estimated regression coefficient was further used to adjust hypothetical Do2 thresholds, and Do2 excursions below the threshold were quantified as magnitude-durations. The lowest threshold that provided magnitude-durations and linked with an increase in lactOFF was used as the lowest suitable (critical) Do2 at 37°C. RESULTS: Overall, 22 896 time points were analysed in 180 neonates. In 40 patients with lactOFF <2.5 mM, Do2 varied by 22.87 (0.70) ml min-1 m-2 °C-1. When varying the Do2 threshold between 340 and 380 ml min-1 m-2, excursions below the threshold were linked with incremental lactOFF. A 100 ml m-2 excursion below the 340 ml min-1 m-2Do2 threshold increased the risk of a 1 mM increment in lactOFF by 22% (odds ratio: 1.22; 95% confidence interval: 1.02-1.45). CONCLUSIONS: It was found that 340 ml min-1 m-2 is likely to represent the lowest suitable Do2 required in neonates to maintain aerobic metabolism during normothermic CPB.

Oxygen impact on genome structure and chemistry / 药学学报

Oxygen is an essential element for life, which is mostly consumed at mitochondria for energy metabolism. For the genome inside nucleus, oxygen conducts structural regulations and chemical modifications through multiple pathways, where reactive oxygen species (ROS) serve as important messenger molecules. The highly activated ROS have the ability to produce different kinds of DNA lesions, while ferrous ions provide supports in many forms. Under the combinatorial action of oxygen and iron, almost all the genomic biochemical processes, such as replication, transcription and DNA damage repair are affected. Moreover, the variation of environmental oxygen concentration, particularly hypoxia that presents in many major diseases and critical physiological stages, provokes the responds at the genomic level. While the factors that lead to these genomic alterations are potential drug targets and deserve systematic investigations, herein, we collect the existing knowledge in the effects of ROS, ferrous ion and cell hypoxia on genome, along with brief discussions of the related drug molecules.

[Human umbilical mesenchymal stem cells-derived exosomes modulate the proliferation, apoptosis and migration of human retinal pigment epithelial cells in hypoxia].

Objective: To study the effects of human umbilical mesenchymal stem cells (HUMSCs) exosomes on the proliferation and apoptotic as well as migration of human retinal pigment epithelial cells (HRPE) in hypoxia, and explore its mechanism. Method: Direct adherent culture was adopted to cultivate umbilical cord mesenchymal stem cells and amplified to the fourth generation. Markers on the cell surface were identified by flow cytometry. Culture medium was collect without serum from the 4th generation umbilical cord mesenchymal stem cells. Exosomes were separated and extracted, then the ultrastructure was observed under electron microscope and examined expression of CD63 and CD9 protein by Western blot method with isolated and extracted exosomes. HRPE was cultivated in vitro culture, proliferation was detected at the time point of 0, 1, 2, 3, 4, 5 d with MTT assay under hypoxic condition. Meanwhile, the cell migration was quantified by Wound-Healing Assay under hypoxic condition at 0, 24, 48 and 72 h respectively combined with apoptosis test. The HRPE cells in the growth period were divided into 5 groups: the control group, the hypoxia group and the pretreated exosomes group (100, 200, 300 µg/ml). In all groups, apoptosis was observed by Annexin V/PI dual-dye flow cytometry after 48 h's incubation. Proliferation was observed by MTT assay and the migration was observed with Wound-Healing Assay. Results: Flow cytometry detection of the surface marker of HUMSCs in the 4th generation showed strong positive expression of CD105, CD73, CD90. It was suggested that HUMSCs with isolated culture had MSC specific phenotype with duction of lipids and osteoblasts in vitro. The separated exosomes were observed with spherical membranous structures in different sizes by scanning electron microscopy, and Western blot detected positive expression of CD63 and CD9. In vitro culture of HRPE detected by MTT assay for cell proliferation at the time of hypoxic 0, 1, 2, 3, 4, 5 d, the results showed that, comparing with time point 0 d, other groups had statistically significant OD values. In the first 2 days, the proliferation ability of RPE cells gradually increased as the time of hypoxia prolonged(1.862±0.135, 2.278±0.244). After 3 d, the proliferation ability of RPE cells gradually decreased(1.419±0.124, 1.599±0.156). Wound-Healing Assay results showed that the migration distance gradually increased as[(29.883±4.504), (36.200±1.928) µm] the time of hypoxia increased from 0 to 72 h. The cells were fully covering at the point of 72 h [(1.223±0.194), (0.430±0.299) µm]. Apoptosis test results showed that the number of apoptotic cells was different(3.628%±1.348%, 20.123%±1.183%) with the extension of hypoxia Oxygen before 2 d from 0 to 72 h. At the time of d3, there were more apoptotic cells(42.290%±3.217%). There is a significant difference from pre-2d.RPE cells were divided into 5 groups: the control group, the hypoxia group and the pretreated exosomes group (100, 200, 300 µg/ml).After 48 h hypoxia incubation, MTT assay results showed that, compared with the control group (1.870±0.499), the number of cell proliferation was significantly increased (t=-3.116, P<0.05), while compared with the hypoxia group(2.616±0.307), the proliferation number of exosomes was significantly reduced [(2.041±0.115), (1.931±0.205), (1.929±0.025); t=-4.920, -4.540, -5.286, P<0.01], and there was no significant difference between groups with different doses of the exosomes (F=1.181,P>0.05). Annexin V/PI dual-dye flow cytometry was used to observe the apoptosis results. Compared with the control group 1.180%±0.689%, the number of apoptosis in hypoxia group was significantly increased (19.273%±1.194%, t=-32.141, P<0.01), while compared with the hypoxia group, the number of apoptosis in the exosomes was significantly decreased (12.318%±1.087%, 11.878%±1.348%, 11.090%±1.716%; t=-10.547, -10.057, 9.589, P<0.01). There was no significant difference between the groups with different doses of exosomes (F=1.173, P>0.05). Wound-Healing Assay results showed that, compared with the control group(68.047±2.851) µm, the migration distance of the hypoxia group was significantly increased [(13.470±2.255)µm, t=36.778, P<0.01] while compared with the hypoxia group, the migration distance of the exosomes was reduced (33.110±1.774, 24.650±1.175, 26.440±1.674; t=11.766, 10.770, 11.311, P<0.01), and there was no significant difference between the groups of the exosomes (F=1.179, P>0.05). Conclusion: Human umbilical cord mesenchymal stem cells can effectively inhibit the apoptosis and migration of HRPE cells in hypoxia. It provides a theoretical basis for the research and treatment of RPE related diseases. (Chin J Ophthalmol, 2019, 55: 933-941).

Hyperoxygenated Hydrogen-Rich Solution Suppresses Lung Injury Induced by Hemorrhagic Shock in Rats.

BACKGROUND: Hemorrhagic shock could induce acute lung injury (ALI), which is associated with cell hypoxia, lung tissue inflammation, free radical damage, and excessive cell apoptosis. Our previous studies demonstrated that hyperoxygenated solution could alleviate cell hypoxia. Furthermore, hydrogen-rich solution (HS) could relieve lung tissue inflammation, free radical damage and excessive cell apoptosis. Therefore we hypothesize that Hyperoxygenated Hydrogen-rich solution (HOHS) can protect the lung against ALI. MATERIALS AND METHODS: SD rats were randomly divided into five groups (n = 6 at each time point in each group) and were exposed to Hemorrhagic shock induced ALI, and then treated with lactated Ringer's solution (LRS), hyperoxygenated solution, HS, and HOHS, respectively. The protective effects of these solutions were assessed using methods as follows: arterial blood samples were collected for blood gas analysis; Bronchoalveolar lavage fluid was collected for cell count and protein quantification; lung tissue samples were collected to measure wet/dry ratio, as well as levels of T-SOD, MDA, TNF-α, and IL-6; Caspase-3 and TUNEL-positive cells, and pathological changes were observed under light microscope; ALI was scored using the Smith scoring method; ultrastructural changes of lung tissues were further observed with transmission electron microscopy. RESULTS: The results indicated that PaO2, PaCO2, and T-SOD increased in the three treatment groups (P < 0.05), most significantly in the HOHS group (P < 0.01) compared with the LRS group; and conversely that the levels of lactate, MDA, TNF-α and IL-6, cell count, protein content, caspase-3 and TUNEL-positive cells as well as ALI score decreased in the three treatment groups (P < 0.05), most significantly in the HOHS group (P < 0.01) compared with the LRS group. Morphological observation with optical microscope and electron microscopy showed that compared with the LRS group, cell damage in the three treatment groups improved to a varying extent, especially evident in the HOHS group. CONCLUSIONS: These findings demonstrate that HOHS can protect the lung against ALI induced by hemorrhagic shock.