Small extracellular vesicles from hypoxia-preconditioned bone marrow mesenchymal stem cells attenuate spinal cord injury via miR-146a-5p-mediated regulation of macrophage polarization

Spinal cord injury is a disabling condition with limited treatment options.Multiple studies have provided evidence suggesting that small extracellular vesicles(SEVs)secreted by bone marrow mesenchymal stem cells(MSCs)help mediate the beneficial effects conferred by MSC transplantation following spinal cord injury.Strikingly,hypoxia-preconditioned bone marrow mesenchymal stem cell-derived SEVs(HSEVs)exhibit increased therapeutic potency.We thus explored the role of HSEVs in macrophage immune regulation after spinal cord injury in rats and their significance in spinal cord repair.SEVs or HSEVs were isolated from bone marrow MSC supernatants by density gradient ultracentrifugation.HSEV administration to rats via tail vein injection after spinal cord injury reduced the lesion area and attenuated spinal cord inflammation.HSEVs regulate macrophage polarization towards the M2 phenotype in vivo and in vitro.Micro RNA sequencing and bioinformatics analyses of SEVs and HSEVs revealed that mi R-146a-5p is a potent mediator of macrophage polarization that targets interleukin-1 receptor-associated kinase 1.Reducing mi R-146a-5p expression in HSEVs partially attenuated macrophage polarization.Our data suggest that HSEVs attenuate spinal cord inflammation and injury in rats by transporting mi R-146a-5p,which alters macrophage polarization.This study provides new insights into the application of HSEVs as a therapeutic tool for spinal cord injury.

Hypoxia and inflammatory factor preconditioning enhances the immunosuppressive properties of human umbilical cord mesenchymal stem cells

BACKGROUND Mesenchymal stem cells(MSCs)have great potential for the treatment of various immune diseases due to their unique immunomodulatory properties.However,MSCs exposed to the harsh inflammatory environment of damaged tissue after intravenous transplantation cannot exert their biological effects,and therefore,their therapeutic efficacy is reduced.In this challenging context,an in vitro preconditioning method is necessary for the development of MSC-based therapies with increased immunomodulatory capacity and transplantation efficacy.AIM To determine whether hypoxia and inflammatory factor preconditioning increases the immunosuppressive properties of MSCs without affecting their biological characteristics.METHODS Umbilical cord MSCs(UC-MSCs)were pretreated with hypoxia(2%O_(2))exposure and inflammatory factors(interleukin-1β,tumor necrosis factor-α,interferon-γ)for 24 h.Flow cytometry,polymerase chain reaction,enzyme-linked immunosorbent assay and other experimental methods were used to evaluate the biological characteristics of pretreated UC-MSCs and to determine whether pretreatment affected the immunosuppressive ability of UC-MSCs in coculture with immune cells.RESULTS Pretreatment with hypoxia and inflammatory factors caused UC-MSCs to be elongated but did not affect their viability,proliferation or size.In addition,pretreatment significantly decreased the expression of coagulationrelated tissue factors but did not affect the expression of other surface markers.Similarly,mitochondrial function and integrity were retained.Although pretreatment promoted UC-MSC apoptosis and senescence,it increased the expression of genes and proteins related to immune regulation.Pretreatment increased peripheral blood mononuclear cell and natural killer(NK)cell proliferation rates and inhibited NK cell-induced toxicity to varying degrees.CONCLUSION In summary,hypoxia and inflammatory factor preconditioning led to higher immunosuppressive effects of MSCs without damaging their biological characteristics.

Desferoxamine preconditioning protects against cerebral ischemia in rats by inducing expressions of hypoxia inducible factor 1α and erythropoietin

Objective To investigate whether desferoxamine （DFO） preconditioning can induce tolerance against cerebral ischemia and its effect on the expression of hypoxia inducible factor 1 α （HIF- 1α） and erythropoietin （EPO） in vivo and in vitro. Methods Rat model of cerebral ischemia was established by middle cerebral artery occlusion with or without DFO administration. Infarct size was examined by TTC staining, and the neurological severity score was evaluated according to published method. Cortical neurons were cultured under ischemia stress which was mimicked by oxygen-glucose deprivation （OGD）, and the neuron damage was assessed by MTT assay. Immunofluorescent staining was employed to detect the expressions of HIF-1 and EPO. Results The protective effect induced by DFO （decreasing the infarction volume and ameliorating the neurological function） appeared at 2 d after administration ofDFO （post-DFO）, lasted until 7 d and disappeared at 14 d （P 〈 0.05）; the most effective action was observed at 3 d post-DFO. DFO induced tolerance of cultured neurons against OGD： neuronal viability was increased 23%, 34%, 40%, 48% and 56% at 8 h, 12 h, 24 h, 36 h, and 48 h, respectively, post-DFO （P 〈 0.05）. Immunofluorescent staining found that HIF-1 α and EPO were upregulated in the neurons of rat brain at 3 d and 7 d post-DFO; increase of HIF-1 α and EPO appeared in cultured cortex neurons at 36 h and 48 h post-DFO. Conclusion DFO induced tolerance against focal cerebral ischemia in rats, and exerted protective effect on OGD cultured cortical neurons. DFO significant induced the expression of HIF- 1 α and EPO both in vivo and in vitro. DFO preconditioning can protect against cerebral ischemia, which may be associated with the synthesis of HIF- 1 α and EPO.

Hypoxia inducible factor-1α mediates protective effects of ischemic preconditioning on ECV-304 endothelial cells

AIM: To investigate whether hypoxia inducible factor-1α (HIF-1α) is linked to the protective effects of ischemic preconditioning (IP) on sinusoidal endothelial cells against ischemia/reperfusion injury. METHODS: Sinusoidal endothelial cell lines ECV-304 were cultured and divided into four groups: control group, cells were cultured in complete DMEM medium; cold anoxia/warm reoxygenation (A/R) group, cells were preserved in a 4℃ UW solution in a mixture of 95% N2 and 5% CO2 for 24 h; anoxia-preconditioning (APC) group, cells were treated with 4 cycles of short anoxia and reoxygenation before prolonged anoxia- preconditioning treatment; and anoxia-preconditioning and hypoxia inducible factor-1α (HIF-1α) inhibitor (I-HIF-1) group, cells were pretreated with 5 μm of HIF-1α inhibitor NS398 in DMEM medium before subjected to the same treatment as group APC. After the anoxia treatment, each group was reoxygenated in a mixture of 95% air and 5% CO2 incubator for 6 h. Cytoprotections were evaluated by cell viabilities from Trypan blue, lactate dehydrogenase (LDH) release rates, and intracellular cell adhesion molecule-1 (ICAM-1) expressions. Expressions of HIF-1α mRNA and HIF-1α protein from each group were determined by the RT-PCR method and Western blotting, respectively. RESULTS: Ischemia preconditioning increased cell viability, and reduced LDH release and ICAM-1 expressions. Ischemia preconditioning also upregulated the HIF-1α mRNA level and HIF-1α protein expression. However, all of these changes were reversed by HIF-1α inhibitor NS398.CONCLUSION: Ischemia preconditioning effectively inhibited cold hypoxia/warm reoxygenation injury to endothelial cells, and the authors showed for the first time HIF-1α is causally linked to the protective effects of ischemic preconditioning on endothelial cells.

Hypoxic Preconditioning Eliminates Differences in the Innate Resistance of Rats to Severe Hypoxia

Hypoxic preconditioning is able to increase the body’s resistance to hypoxic/ischemic stress. Understanding how to apply the hypoxic response to initiate the protective mechanism of ischemic preconditioning is a high priority. However, the relationship between innate resistance to hypoxic stress and preconditioning efficiency of moderate hypoxia has been poorly studied. In our work, the efficiency of single moderate hypobaric hypoxia (HBH) for resistance to severe hypobaric hypoxia (SHBH) was studied on intact rats and those pre-tested under SHBH with low, intermediate and high resistance to hypoxia. HBH has a significant preconditioning action on the resistance to hypoxia over a wide range from 270 to 1464 s (4.5 to 24.5 min) and at the same time eliminates the differences in the endurance under SHBH between all rat groups. It is concluded that 1) HBH preconditioning efficiency does not depend on an innate resistance to SHBH and prior hypoxic experience of rats;and 2) the pretesting to severe hypoxia has no value for predicting the hypoxic preconditioning efficiency and study of adaptive mechanisms.

Searching for the optimal precondition procedure for mesenchymal stem/stromal cell treatment:Facts and perspectives

Mesenchymal stem/stromal cells are potential optimal cell sources for stem cell therapies,and pretreatment has proven to enhance cell vitality and function.In a recent publication,Li et al explored a new combination of pretreatment condi-tions.Here,we present an editorial to comment on their work and provide our view on mesenchymal stem/stromal cell precondition.

Molecular mechanisms of liver preconditioning

Ischemia/reperfusion (I/R) injury still represents an important cause of morbidity following hepatic surgery and limits the use of marginal livers in hepatic transplantation. Transient blood flow interruption followed by reperfusion protects tissues against damage induced by subsequent I/R. This process known as ischemic pre-conditioning (IP) depends upon intrinsic cytoprotective systems whose activation can inhibit the progression of irreversible tissue damage. Compared to other organs,liver IP has additional features as it reduces inflammation and promotes hepatic regeneration. Our present understanding of the molecular mechanisms involved in liver IP is still largely incomplete. Experimental studies have shown that the protective effects of liver IP are triggered by the release of adenosine and nitric oxide and the subsequent activation of signal networks involving protein kinases such as phosphatidylinositol 3-kinase,protein kinase C δ/ε and p38 MAP kinase,and transcription factors such as signal transducer and activator of transcription 3,nuclear factor-κB and hypoxia-inducible factor 1. This article offers an overview of the molecular events underlying the preconditioning effects in the liver and points to the possibility of developing pharmacological approaches aimed at activating the intrinsic protective systems in patients undergoing liver surgery.

Acupuncture preconditioning protects hippocampal neurons from transient ischemia/reperfusion injury

The present study established a model of brain ischemia in aged rats using four-vessel occlusion.We observed hippocampal CA1 neuronal apoptosis and apoptosis-mediated protease caspase-3 expression following preconditioning of electroacupuncture at Baihui（GV 20）.Our results showed that the number of hippocampal CA1 normal neurons was decreased,and degenerated neurons were increased 12 hours to 3 days following cerebral ischemia/reperfusion.The number of hippocampal CA1 apoptotic neurons and caspase-3-positive neurons in rats with cerebral ischemia/reperfusion injury was significantly decreased following acupuncture preconditioning.Acupuncture preconditioning protects aged rats against ischemia/reperfusion injury by regulating caspase-3 protein expression.

Extracellular vesicles from hypoxia-preconditioned mesenchymal stem cells alleviates myocardial injury by targeting thioredoxininteracting protein-mediated hypoxia-inducible factor-1αpathway

BACKGROUND Extracellular vesicles(EVs)derived from hypoxia-preconditioned(HP)mesenchymal stem cells(MSCs)have better cardioprotective effects against myocardial infarction(MI)in the early stage than EVs isolated from normoxic(NC)-MSCs.However,the cardioprotective mechanisms of HP-EVs are not fully understood.AIM To explore the cardioprotective mechanism of EVs derived from HP MSCs.METHODS We evaluated the cardioprotective effects of HP-EVs or NC-EVs from mouse adipose-derived MSCs(ADSCs)following hypoxia in vitro or MI in vivo,in order to improve the survival of cardiomyocytes(CMs)and restore cardiac function.The degree of CM apoptosis in each group was assessed by the terminal deoxynucleotidyl transferase dUTP nick end-labeling and Annexin V/PI assays.MicroRNA(miRNA)sequencing was used to investigate the functional RNA diversity between HP-EVs and NC-EVs from mouse ADSCs.The molecular mechanism of EVs in mediating thioredoxin-interacting protein(TXNIP)was verified by the dual-luciferase reporter assay.Co-immunoprecipitation,western blotting,and immunofluorescence were performed to determine if TXNIP is involved in hypoxia-inducible factor-1 alpha(HIF-1α)ubiquitination and degradation via the chromosomal region maintenance-1(CRM-1)-dependent nuclear transport pathway.RESULTS HP-EVs derived from MSCs reduced both infarct size(necrosis area)and apoptotic degree to a greater extent than NC-EVs from CMs subjected to hypoxia in vitro and mice with MI in vivo.Sequencing of EV-associated miRNAs showed the upregulation of 10 miRNAs predicted to bind TXNIP,an oxidative stress-associated protein.We showed miRNA224-5p,the most upregulated miRNA in HP-EVs,directly combined the 3’untranslated region of TXNIP and demonstrated its critical protective role against hypoxia-mediated CM injury.Our results demonstrated that MI triggered TXNIP-mediated HIF-1αubiquitination and degradation in the CRM-1-mediated nuclear transport pathway in CMs,which led to aggravated injury and hypoxia tolerance in CMs in the early stage of MI.CONCLUSION The anti-apoptotic effects of HP-EVs in alleviating MI and the hypoxic conditions of CMs until reperfusion therapy may partly result from EV miR-224-5p targeting TXNIP.

Hypoxic preconditioning：effect,mechanism and clinical implication(Part 1)

Hypoxic preconditioning(HPC) refers to exposure of organisms,systems,organs,tissues or cells to moderate hypoxia/ischemia that is able to result in a resistance to subsequent severe hypoxia/ischemia in tissues and cells.The effects exerted by HPC are well documented.The original local in situ(LiHPC) is now broadened to remote ectopic organs-tissues(ReHPC) and extended crossly to cross pluripotential HPC(CpHPC) induced by a variety of stresses other than hypoxia/ischemia,including cancer,for example.We developed a unique animal model of repetitive autohypoxia in adult mice,and studied systematically on the effects and mechanisms of HPC on the model in our laboratory since the early 1960 s.The tolerances to hypoxia and protection from injury increased significantly in this model.The adult mice behave like hypoxia-intolerant mammalian newborns and hypoxia-tolerant adult animals during their exposure to repetitive autohypoxia.The overall energy supply and demand decreased,the microorganization of the brain maintained and the spacial learning and memory ability improved but not impaired,the detrimental neurochemicals such as free radicals down-regulated and the beneficial neurochemicals such as adenosine(ADO) and antihypoxic gene(s)/factor(s)(AHGs/AHFs) up-regulated.Accordingly,we hypothesize that mechanisms for the tolerance / protective effects of HPC are fundamentally depending on energy saving and brain plasticity in particular.It is thought that these two major mechanisms are triggered by exposure to hypoxia/ischemia via oxygen sensing-transduction pathways and HIF-1 initiation cascades.We suggest that HPC is an intrinsic mechanism developed in biological evolution and is a novel potential strategy for fighting against hypoxia-ischemia and other stresses.Motivation of endogenous antihypoxic potential,activation of oxygen sensing- signal transduction systems and supplement of exogenous antihypoxic substances as well as development of HPC appliances and HPC medicines such as AHFs are encouraged based on our basic research on HPC.HPC may result in therapeutic augmentation of the endogenous cytoprotection in hypoxic-ischemic or suffering from other diseases' patients.Evolutionary consideration of HPC and clinical implications of HPC are both discussed to guide future research.The product of AHF is expected to be one of the most effective first aid medicines to rescue patients in critical condition.HPC is beginning to be used in surgery and is expected to be developed into a feasible adaptive medicine in the near future.

Hypoxic/ischemic preconditioning attenuate PKCδ-medi-ated injury in patients and mice with cerebral infarction

Objective:cerebral ischemic/hypox-ic preconditioning(I/HPC)is an endogenous strategy in which brief periods of sublethal ischemia/hypoxia render neural tissues resistant to subsequent ischemic/hypoxic damage.This phenomenon has been found in the brain,heart,liver,intestine,muscle,kidneys,and lung.How-ever,whether HPC has a protective effect on secondary cerebral ischemic injury or protein kinase Cδ(PKCδ)within ischemic patients and animal models is still un-clear.Methods:using a hypoxic preconditioned mouse model and a middle cerebral artery occlusion mouse mod-el,combined with 2,3,5-triphenyl tetrazolium chloride(TTC)staining,SDS-polyacrylamide gel electrophoresis(SDS-PAGE),and Western blot,we observed changes in infarction size,density,edema ratio,and changes in PKCδand membrane translocation within the ischemic cortex of the middle cerebral artery occlusion(MCAO)mice.Results:HPC can attenuate neurological deficits and cerebral ischemic injuries of mice following MCAO,including decreases in infarct size,edema ratio,densities of infarct area,and neuron loss.In addition,HPC inhib-its PKCδmembrane translocation in the penumbra of the MCAO-induced ischemic cortex.We found that admin-istration of PKCδ-specific inhibitor dV1-1 mimics the neuroprotective effects of HPC,and nonisoform-specif-ic activation of PKC can partially abolish HPC-induced neuroprotection.Ischemic preconditioning decreased the levels of PKCδin the serum of patients with cerebral in-farction and reduced the cerebral nerve damage caused by ischemia.Conclusion:hypoxic/ischemic precondi-tioning attenuates PKCδ-mediated injury in patients and mice.These findings enrich our understanding of the sig-nal transduction mechanism underlying cerebral HPC and provide clues to developing medicine against ischemia/hypoxia-induced cerebral injuries.

外泌体及预处理方式对牙髓再生的作用

背景:现有研究已经证实外泌体可有效促进牙髓再生,而经预处理来源的外泌体其生物学功能和特性会发生显著改变,对细胞的增殖、迁移和成牙分化产生不同的影响。目的:探讨外泌体及其预处理方式在牙髓再生领域的应用现状,归纳和总结影响外泌体发挥作用的预处理方式,并阐述外泌体及其预处理方式对牙髓再生的作用。方法:检索万方、中国知网、PubMed和Web of Science数据库中2006-2022年发表的相关文献,以“外泌体,牙髓再生,预处理方式”等为中文检索词,以“Exosomes,Pulp regeneration,Preconditioning method”等为英文检索词进行检索,共纳入78篇文献进行综述分析。结果与结论:①外泌体具有良好的生物相容性、低免疫原性和无细胞毒性等优势,可以通过促进干细胞成牙、成神经和成血管化进而诱导牙髓组织的新生。②经预处理衍生的外泌体可以增强对组织的修复和再生能力,并对再生牙髓的质量有显著影响。③目前应用在牙髓再生领域中的预处理方式包括炎症刺激、低氧诱导、条件培养基和三维培养,其分泌的外泌体均能有效改善再生牙髓的质量,但是不同的预处理方式对牙髓再生的具体效果和机制在未来尚需探索。

低氧预适应对小鼠自然杀伤细胞活性的影响

目的:探讨低氧预适应(hypoxic preconditioning,HPC)条件下对小鼠自然杀伤细胞(natural killer cell,NK)杀伤活性与功能的影响。方法:将小鼠分对照组(A组)、急性低氧组(B组)和HPC组(C组)3组,通过MTT比色法分别检测3组小鼠NK细胞的杀伤活性,通过EILSA检测NK细胞分泌干扰素-γ,Real-time PCR检测穿孔素、颗粒酶的表达量,并采用SPSS 19.0统计学软件进行数据分析。结果:低氧预适应组与急性低氧组相比NK杀伤力较强(P<0.05),其中12 h时NK细胞活性明显增强(P<0.05)。小鼠NK细胞分泌IFN-γ从4 h到16 h均逐渐下降(P<0.05),显示低氧预适应组随着时间的延长较低氧组更能适应低氧;HPC组与B组相比低氧损伤减轻,分泌穿孔素、颗粒酶表达多于低氧组(P<0.05)。结论:初步确定NK细胞在HPC与低氧组相比,NK细胞杀伤功能有所恢复;IFN-γ、穿孔素、颗粒酶蛋白的分泌量均有所升高。

远隔缺血预处理分离液对人肾小管上皮细胞缺氧性损伤的保护作用

目的:研究远隔缺血预处理(RIPC)分离液对人肾小管上皮细胞(HK-2)缺氧/复氧(H/R)损伤的影响及机制。方法:将HK-2细胞分为时间对照组、H/R、RIPC处理组、自噬干预组。时间对照组细胞在正常条件下培养,RIPC组细胞使用RIPC分离液预处理,自噬干预组细胞在RIPC处理基础上,加入自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3MA),后三组细胞使用三气培养箱进行H/R构建损伤模型。模型成功后使用光学显微镜观察各组细胞状态,应用Western blot观察LC3Ⅱ/Ⅰ蛋白、Beclin1蛋白的表达水平。结果:镜下示RIPC分离液可明显减轻H/R对HK-2细胞的损伤,加入自噬抑制剂,RIPC分离液对HK-2细胞H/R损伤的保护作用被阻断。Western blot结果显示,RIPC分离液可使HK-2细胞线粒体中的自噬特异性蛋白LC3Ⅱ/Ⅰ、Beclin1表达水平增高。Real-time PCR结果显示,与H/R组比,RIPC组LC3及Beclin1的mRNA表达量明显升高;加入自噬抑制剂,HK-2细胞线粒体中的自噬特异性蛋白LC3Ⅱ/Ⅰ、Beclin1表达降低,LC3及Beclin1的mRNA表达量同时也明显降低。结论:RIPC分离液对HK-2细胞H/R后损伤具有保护作用,作用机制可能与线粒体自噬相关。

cPKCγ在HPC抗N2a细胞OGD损伤中作用及其分子机制

目的在离体细胞水平,探讨经典型蛋白激酶Cγ(cPKCγ)在低氧预适应(HPC)对抗小鼠成神经瘤细胞(N2a)氧糖剥夺(OGD)损伤中作用及其可能的细胞分子机制。方法建立N2a细胞HPC和OGD模型,用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)比色法、乳酸脱氢酶(LDH)漏出率、原位末端标记(TUNEL)和蛋白印迹(Western blot)几种方法,分别检测N2a细胞损伤、坏死、凋亡和自噬水平。结果 OGD 2和4 h可使N2a细胞的生存率显著降低、死亡率明显增高(P<0.05,n=6);HPC(20 min)明显改善OGD 3 h对N2a细胞的损伤作用,而cPKCγ抑制剂Go6983(6 nmol/L)则显著解除HPC对OGD细胞的保护作用(P<0.05,n=16);OGD 3 h明显增加N2a细胞的凋亡数目(P<0.05,n=6),但HPC和Go6983+HPC均不能明显影响OGD细胞凋亡水平;OGD 3 h显著提高N2a细胞的自噬水平,Go6983和HPC+Go6983均可明显降低OGD细胞的自噬水平(P<0.05,n=6),而HPC则对OGD细胞的自噬水平无明显影响;HPC明显改善OGD 3 h致N2a细胞坏死的程度,而Go6983则可解除HPC的保护作用(P<0.05,n=16)。结论在离体细胞水平证实了cPKCγ在HPC改善N2a细胞OGD损伤中的作用,且这种作用主要与降低OGD细胞的坏死水平有关。

Hypoxia inducible factor 1α promotes interleukin-1 receptor antagonist expression during hepatic ischemia-reperfusion injury

BACKGROUND Ischemia-reperfusion injury(IRI) is a major risk associated with liver surgery and transplantation,and its pathological mechanism is complex.Interleukin-1 receptor antagonist(IL-1ra) can protect the liver from IRI.However,the regulatory mechanism of IL-1ra expression is still unclear.AIM To identify the mechanism that could protect the liver in the early stage of IRI.METHODS To screen the key genes in hepatic IRI,we performed RNA sequencing and gene enrichment analysis on liver tissue from mice with hepatic IRI.Subsequently,we verified the expression and effect of IL-1ra in hepatic IRI.We also used promoter mutagenesis and chromatin immunoprecipitation assay to search for the transcriptional regulatory sites of hypoxia-inducible factor(HIF)-1α.Finally,to explore the protective mechanism of ischemic preconditioning(IP),we examined the expression of HIF-1α and IL-1ra after IP.RESULTS We identified IL-1ra as a key regulator in hepatic IRI.The expression of IL-1ra was significantly upregulated after hepatic IRI both in vivo and in vitro.Furthermore,we found that HIF-1αregulated Il-1ra transcription in response to hypoxia.Increased HIF-1α accumulation promoted IL-1ra expression,whereas inhibition of HIF-1α exhibited the opposite effect.We also confirmed a predominant role for hypoxia response element in the regulation of Il1ra transcription by HIF-1αactivation.Of note,we demonstrated that IP protects against hepatic IRI by inducing IL-1ra expression,which is mediated through HIF-1α.CONCLUSION We demonstrated that ischemia or hypoxia leads to increased expression of IL-1ra through HIF-1α.Importantly,IP protects the liver from IRI via the HIF-1α–IL-1ra pathway.

Correlation of hypoxia-inducible factor-1 alpha and erythropoietin protein and mRNA to cerebral ischemic tolerance in a focal ischemia/reperfusion model using the twice suture method

BACKGROUND： Numerous studies have shown that transient ischemic preconditioning induces cerebral ischemic tolerance. However, the underlying mechanisms of endogenous protection following ischemic preconditioning remain unclear. OBJECTIVE： To dynamically measure erythropoietin and hypoxia-inducible factor-1α （HIF-1α） mRNA and protein expression at various times following preconditioning, and to investigate effects of erythropoietin and HIF-1α on cerebral ischemic tolerance in a model of focal ischemia/reperfusion established using the twice suture method. DESIGN, TIME AND SETTING： The randomized, controlled study was performed at the Institute of Anatomy, Medical College, Qingdao University, China from March 2006 to March 2007. MATERIALS： Rabbit anti-rat HIF-1α monoclonal antibody and biotinylated goat anti-rabbit IgG （Boster, China）, rabbit anti-rat erythropoietin monoclonal antibody （Santa Cruz Biotechnology, USA）, and one-step RT-PCR kit （Qiagen, Germany） were used in this study. METHODS： A total of 99 healthy, male, Wistar rats were randomly assigned to three groups： sham surgery （n = 9）, non-ischemic preconditioning （n = 45）, and ischemic preconditioning （n = 45）. In the ischemic preconditioning group, rat models of pre-ischemia-reperfusion-ischemia-reperfusion were established by occluding the left middle cerebral artery using the twice suture method. In the non-ischemic preconditioning group, pre-ischemia was replaced by sham surgery. Subsequently, the ischemic preconditioning and non-ischemic preconditioning groups were equally divided into five subgroups according to time of first reperfusion, including 1-, 3-, 7-, 14-, and 21-day subgroups. The sham surgery group received the sham surgery twice. MAIN OUTCOME MEASURES： HIF-la and erythropoietin protein expression was measured in the cerebral cortex, corpus striatum, and hippocampus of the ischemic hemisphere. HIF-1α and erythropoietin mRNA expression were determined in the frontal and parietal cortex of the ischemic hemisphere. RESULTS： （1） Intergroup comparison： compared with the non-ischemic preconditioning group, HIF-1α protein expression significantly increased in the rat cerebral cortex, corpus striatum, and hippocampus in the ischemic hemisphere at 1,3, and 7 days following reperfusion in the ischemic preconditioning group （P 〈 0.05 or P 〈 0.01）. Erythropoietin protein expression significantly increased in the cerebral cortex, corpus striatum, and hippocampus, as well as HIF-1α and erythropoietin mRNA expression in the frontal and parietal cortex in the ischemic hemisphere, at 3 and 7 days following reperfusion in the ischemic preconditioning group （P 〈 0.05）. （2） Temporal expression： HIF-1α protein expression in the rat cerebral cortex, corpus striatum, and hippocampus, as well as HIF-la mRNA expression in the frontal and parietal cortex, in the ischemic hemisphere increased at 3 days, and gradually decreased from 7 days following reperfusion in the ischemic preconditioning group. Temporal erythropoietin protein and mRNA expression was consistent with HIF-1α protein expression. （3） Correlation： erythropoietin mRNA expression positively correlated with HIF-1α mRNA expression （r= 0.737, P 〈 0.01）. CONCLUSION： Ischemic preconditioning induced cerebral ischemic tolerance. Pre-ischemiainduced increase in endogenous HIF-1αexpression, as well as its target gene erythropoietin, participated in the formation of cerebral ischemic tolerance.

低氧预适应对魁蚶在低氧胁迫下生理生化指标的影响

本研究以未进行低氧预适应的魁蚶(Scapharcabroughtonii)为对照组(C组),分析了2次低氧预适应(H2组)和4次低氧预适应(H4组)的魁蚶在溶解氧(DO)约为2.0mg/L低氧胁迫48h内的摄食、呼吸代谢和酶活力的变化规律。结果显示,3组魁蚶的摄食率(IR)在胁迫初期急剧下降,后期均随时间的延长逐渐恢复,至48 h时,H组恢复程度显著高于C组(P<0.05);C组、H2组和H4组魁蚶的耗氧率(OR)随时间变化呈逐渐升高的趋势,48 h比0 h分别提高了1.15、1.08、0.73倍;3组排氨率(NR)表现出不同的变化趋势,至48h时,C组、H2组和H4组分别为0h的1.67、1.30、0.97倍;C组的氧氮比(O/N)相对平稳,H组的变化范围相对较大。3组的细胞色素C氧化酶(COX)随着低氧胁迫时间的延长呈逐渐降低的趋势,乳酸脱氢酶(LDH)活力和还原型谷胱甘肽酶(GSH)含量整体呈上升趋势,与对照组相比,预低氧组的酶活力在低氧胁迫期间变化相对平稳,应激反应小。研究表明,魁蚶经低氧预适应后再次受到低氧胁迫时,IR升高,OR降低,酶活性相对稳定,低氧预适应能提高魁蚶的耐低氧能力。本研究丰富了魁蚶低氧耐受相关研究的基础数据,为进一步探讨魁蚶低氧耐受机制和创制耐低氧新种质提供了参考资料。

缺氧预处理通过缺氧诱导因子-1α/基质细胞衍生因子-1通路对大鼠血液学相关指标的影响

目的探讨缺氧诱导因子-1α(HIF-1α)/基质细胞衍生因子-1(SDF-1)通路对缺氧预处理大鼠模型的作用。方法76只成年雄性SD大鼠,通过在低压氧舱(海拔5000 m)和西宁市(海拔2260 m)饲养建立缺氧预处理动物模型,随机分为6组:对照组(Ctrl组),高海拔缺氧预处理1 d组(HHP-1d),高海拔缺氧预处理4 d组(HHP-4d)、高海拔缺氧预处理15 d组(HHP-15d),高海拔缺氧预处理30 d组(HHP-30d),中海拔缺氧预处理组(MHP),其中Ctrl组、HHP-4d组、HHP-30d组与MHP组利用7.0 T小动物MRI,T2加权像(T2WI)观察颅内结构及基底动脉直径,连续性自旋动脉标记(ASL)观察海马区及脑干区脑血流,检测各组大鼠血常规,ELISA检测各组大鼠血清HIF-1α和SDF-1浓度及血浆血小板活化因子(PAF)及P-选择素(SELP)浓度。结果缺氧预处理条件下,颅内结构未见明显异常;基底动脉直径增宽,但无统计学意义;脑干区及海马区脑血流量明显升高,差异具有统计学意义(P<0.05),红细胞及白细胞升高,血小板下降,差异具有统计学意义(P<0.05),其中MHP组红细胞和血小板接近Ctrl组;缺氧预处理组HIF-1α浓度明显升高,SDF-1浓度(除HHP-1d组外)明显升高,HHP-1d、HHP-15d组PAF、SELP浓度明显升高,HHP-4d、HHP-30d组PAF浓度明显下降,HHP-4d组SELP浓度明显下降,差异均具有统计学意义(P<0.05)。结论缺氧预处理可通过HIF-1α/SDF-1通路增加机体氧储备量及免疫防御力,提高脑储备能力并发挥脑保护作用,其中以中海拔(海拔2260 m)饲养30 d预处理效果最佳。

Secretive derived from hypoxia preconditioned mesenchymal stem cells promote cartilage regeneration and mitigate joint inflammation via extracellular vesicles

Secretome derived from mesenchymal stem cells (MSCs) have profound effects on tissue regeneration, which could become the basis of future MSCs therapies. Hypoxia, as the physiologic environment of MSCs, has great potential to enhance MSCs paracrine therapeutic effect. In our study, the paracrine effects of secretome derived from MSCs preconditioned in normoxia and hypoxia was compared through both in vitro functional assays and an in vivo rat osteochondral defect model. Specifically, the paracrine effect of total EVs were compared to that of soluble factors to characterize the predominant active components in the hypoxic secretome. We demonstrated that hypoxia conditioned medium, as well as the corresponding EVs, at a relatively low dosage, were efficient in promoting the repair of critical-sized osteochondral defects and mitigated the joint inflammation in a rat osteochondral defect model, relative to their normoxia counterpart. In vitro functional test shows enhancement through chondrocyte proliferation, migration, and matrix deposition, while inhibit IL-1β-induced chondrocytes senescence, inflammation, matrix degradation, and pro-inflammatory macrophage activity. Multiple functional proteins, as well as a change in EVs’ size profile, with enrichment of specific EV-miRNAs were detected with hypoxia preconditioning, implicating complex molecular pathways involved in hypoxia pre-conditioned MSCs secretome generated cartilage regeneration.