Hypoxia-preconditioned bone marrow-derived mesenchymal stem cells protect neurons from cardiac arrest-induced pyroptosis

Cardiac arrest can lead to severe neurological impairment as a result of inflammation,mitochondrial dysfunction,and post-cardiopulmonary resuscitation neurological damage.Hypoxic preconditioning has been shown to improve migration and survival of bone marrow–derived mesenchymal stem cells and reduce pyroptosis after cardiac arrest,but the specific mechanisms by which hypoxia-preconditioned bone marrow–derived mesenchymal stem cells protect against brain injury after cardiac arrest are unknown.To this end,we established an in vitro co-culture model of bone marrow–derived mesenchymal stem cells and oxygen–glucose deprived primary neurons and found that hypoxic preconditioning enhanced the protective effect of bone marrow stromal stem cells against neuronal pyroptosis,possibly through inhibition of the MAPK and nuclear factor κB pathways.Subsequently,we transplanted hypoxia-preconditioned bone marrow–derived mesenchymal stem cells into the lateral ventricle after the return of spontaneous circulation in an 8-minute cardiac arrest rat model induced by asphyxia.The results showed that hypoxia-preconditioned bone marrow–derived mesenchymal stem cells significantly reduced cardiac arrest–induced neuronal pyroptosis,oxidative stress,and mitochondrial damage,whereas knockdown of the liver isoform of phosphofructokinase in bone marrow–derived mesenchymal stem cells inhibited these effects.To conclude,hypoxia-preconditioned bone marrow–derived mesenchymal stem cells offer a promising therapeutic approach for neuronal injury following cardiac arrest,and their beneficial effects are potentially associated with increased expression of the liver isoform of phosphofructokinase following hypoxic preconditioning.

Hypoxic preconditioning stimulates angiogenesis in ischemic penumbra after acute cerebral infarction

Previous studies have demonstrated the protective effect of hypoxic preconditioning on acute cerebral infarction, but the mechanisms underlying this protection remain unclear. To investigate the protective mechanisms of hypoxic preconditioning in relation to its effects on angiogenesis, we in- duced a photochemical model of cerebral infarction in an inbred line of mice （BALB/c）. Mice were then exposed to hypoxic preconditioning 30 minutes prior to model establishment. Results showed significantly increased vascular endothelial growth factor and CD31 expression in the ischemic penumbra at 24 and 72 hours post infarction, mainly in neurons and vascular endothelial cells. Hypoxic preconditioning increased vascular endothelial growth factor and CD31 expression in the ischemic penumbra and the expression of vascular endothelial growth factor was positively related to that of CD31. Moreover, hypoxic preconditioning reduced the infarct volume and improved neu- rological function in mice. These findings indicate that the protective role of hypoxic preconditioning in acute cerebral infarction may possibly be due to an increase in expression of vascular endothelial growth factor and CD31 in the ischemic penumbra, which promoted angiogenesis.

Impact of hypoxic preconditioning on apoptosis and its possible mechanism in orthotopic liver autotransplantation in rats

BACKGROUND: Hepatocyte apoptosis is a severe form of cell death after hepatic ischemia-reperfusion injury (HIRI), and its relief is an important issue in liver transplantation. Hypoxic preconditioning (HP) is considered to have protective effects on HIRI. This study was designed to explore the impact of HP on apoptosis and its possible mechanism during orthotopic liver autotransplantation. METHODS: A modified orthotopic liver autotransplantation model was used to simulate HIRI. Sprague-Dawley rats were randomly divided into normal control, autotransplantation (AT) and HP groups. The HP group was subjected to an 8% oxygen atmosphere for 90 minutes before surgery. At 1, 6 and 24 hours after surgery, the rats were killed and their liver tissue was sampled to assess the expression of Bcl-2 protein. The samples were subjected to blood chemistry study, morphological study under a light or transmission electron microscope, and quantitative study of mitochondria. RESULTS: The serum levels of ALT and AST in the HP group were lower than those in the AT group at 1, 6 and 24 hours after orthotopic liver autotransplantation (P < 0.05). Bcl-2 protein expression was increased in the HP group at each measurement point (P < 0.05). Light microscopy showed that hepatic injury in the AT group was much more severe than in the HP group. Hepatocytes in the AT group showed typical apoptosis signs under a transmission electron microscope. The ultrastructural appearance of hepatocytes in the HP group was much better than in the AT group, and the area, perimeter and diameter of the mitochondria were smaller in the HP group than in the AT group (P < 0.05). CONCLUSIONS: Hepatocytes sense and respond to decreased tissue oxygenation. Stimulation by HP relieves apoptosis by upregulating expression of Bcl-2 protein and its protection of mitochondria after orthotopic liver autotransplantation.

Effect of Hypoxic Preconditioning on Neural Cell Apoptosis and Expression of Bcl-2 and Bax in Cerebral Ischemia-Reperfusion in Rats

In order to investigate the protective effect of hypoxic preconditioning on the cerebral ischemia-reperfusion injury, the expression of Bcl-2 and Bax was detected by using immunohistochemical staining after 3 h cerebral ischemia followed by 1, 6, 12, 24 and 48 h reperfusion respectively in rats treated with or without hypoxic preconditioning before cerebral ischemia. In addition, the apoptosis of neural cells and the behavioral scores for neurological functions recovery were evaluated by TUNEL staining and ＂crawling method＂, respectively. Compared with control group （cerebral ischemia-reperfusion without hypoxic preconditioning）, the expression of Bcl-2 was significantly increased, but that of Bax decreased in the hypoxic preconditioning group （cerebral ischemiareperfusion with hypoxie preconditioning）, both P〈0.05. The pre-treatment with hypoxic preconditioning could reduce the apoptosis of neural cells and promote the neurological function recovery as compared to control group. It was suggested that hypoxic preconditioning may have protective effects on the cerebral ischemia-reperfusion injury by inhibiting the apoptosis of neural cells, increase the expression of Bcl-2 and decrease the expression of Bax.

Hypoxic preconditioning reduces NLRP3 inflammasome expression and protects against cerebral ischemia/reperfusion injury

Hypoxic preconditioning can protect against cerebral ischemia/reperfusion injury. However, the underlying mechanisms that mediate this effect are not completely clear. In this study, mice were pretreated with continuous, intermittent hypoxic preconditioning;1 hour later, cerebral ischemia/reperfusion models were generated by middle cerebral artery occlusion and reperfusion. Compared with control mice, mice with cerebral ischemia/reperfusion injury showed increased Bederson neurological function scores, significantly increased cerebral infarction volume, obvious pathological damage to the hippocampus, significantly increased apoptosis;upregulated interleukin-1β, interleukin-6, and interleukin-8 levels in brain tissue;and increased expression levels of NOD-like receptor family pyrin domain containing 3(NLRP3), NLRP inflammasome-related protein caspase-1, and gasdermin D. However, hypoxic preconditioning significantly inhibited the above phenomena. Taken together, these data suggest that hypoxic preconditioning mitigates cerebral ischemia/reperfusion injury in mice by reducing NLRP3 inflammasome expression. This study was approved by the Medical Ethics Committee of the Fourth Hospital of Baotou, China(approval No. DWLL2019001) in November 2019.

Gene expression changes after hypoxic preconditioning in rat hepatocytes

BACKGROUND: Hypoxic preconditioning can protect hepatocytes against hypoxic injury, but its mechanism has not been elucidated. The aim of this study was to profile gene expression patterns involved in hypoxic preconditioning and probable mechanism at the level of gene expression. METHODS: Hepatocytes were divided into 2 groups: control group and hypoxic preconditioning group. Biotinlabeled cRNA from the control group and the hypoxic preconditioning group was hybridized by oligonucleotide microarray. Genes that were significantly associated with hypoxic preconditioning were filtered, and validated at the level of transcript expression. RESULTS: Forty-three genes with significantly altered expression patterns were discovered and most of them had not been previously reported. Among these genes,genes encoding superoxide dismutase 2 (SOD2)and interleukin 10 (IL-10) in the hypoxic preconditioning group were confirmed to be up-regulated with real-time quantitative PCR. CONCLUSIONS: Many cytokines are involved in hypoxic preconditioning and protect hepatocytes from hypoxiareoxygenation injury, and the increase of oxygen freeradical scavengers and anti-inflammatory factors may play a key role in this phenomenon. Diverse signal pathways are probably involved.

Cerebrospinal fluid from rats given hypoxic preconditioning protects neurons from oxygen-glucose deprivation-induced injury

Hypoxic preconditioning activates endogenous mechanisms that protect against cerebral isch- emic and hypoxic injury. To better understand these protective mechanisms, adult rats were housed in a hypoxic environment （8% 02/92% N2） for 3 hours, and then in a normal oxygen environment for 12 hours. Their cerebrospinal fluid was obtained to culture cortical neurons from newborn rats for 1 day, and then the neurons were exposed to oxygen-glucose deprivation for 1.5 hours. The cerebrospinal fluid from rats subjected to hypoxic preconditioning reduced oxygen-glucose deprivation-induced injury, increased survival rate, upregulated Bcl-2 expression and downregulated Bax expression in the cultured cortical neurons, compared with control. These results indicate that cerebrospinal fluid from rats given hypoxic preconditioning protects against oxygen-glucose deprivation-induced injury by affecting apoptosis-related protein expres- sion in neurons from newborn rats.

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 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.

Hypoxic Preconditioning Improved Neuroprotective Effect of Bone Marrow-Mesenchymal Stem Cells Transplantation in Acute Glaucoma Models

This study explored the novel strategy of hypoxic preconditioning of Bone Marrow Mesenchymal Stem Cells (BM-MSCs) before intra vitreal transplantation to improve neuroprotective effects of Retinal Ganglion Cells (RGCs) in Acute Glaucoma Models. The methods of this research were isolated mesenchymal stem cells from the bone marrow of adult wild-type Sprague-Dawley (SD) rats. BM-MSCs were cultured under normoxic or hypoxic (1% oxygen for 24 hours) conditions. Normoxic or hypoxic BM-MSCs were transplanted intravitreally 1 week after ocular hypertension induction by acutely increasing IOP to 100 - 120 mmHg for 60 minutes. Rats were killed 4 weeks after transplanted. Apoptosis was examined by tunnel assay and expression Brn3b (Brn3b = RGCs marker) by immunohistochemical analysis of the retina. Results showed that transplantation of hypoxic preconditioning BM-MSCs in acute glaucoma models resulted in a significant apoptosis decreasing (p < 0.05) and an significant increasing in RGCs (p < 0.05), as well as enhanced mor-phologic and functional benefits of stem cell therapy versus normoxic BM-MSCs transplantation. Conclusions: Hypoxic preconditioning enhances the capacity of BM-MSCs transplantation to improve neuroprotective effects of RGCs in Acute Glaucoma Models.

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.

Effects of hypoxic preconditioning on myocardial mitochondrial energy metabolism during acute hypoxia in rats

The effects of hypoxic preconditioning (HP) on the functions of myocardial mitochondria and ATP content were studiedin the rat. Rats were exposed to a simulated altitude of 4000m (with a barometric pressure of 43 kPa) for 1d. The myocardialmitochondrial respiratory function was determined with the Clark type O2 electrode, mitochondrial membrane fluidity (MMF) wasassayed with fluorescence polarizative method, and the myocardial content of ATP, ADP and AMP were measured with high performance liquid chromatography. It was found that after the administration of HP, the ATP content was increased from 31.89±2.42/mg·g-1 to 60. 55±3.52/mg·g-1 (P<0.01 ), mitochondrial respiratory control rate (RGR) was increased from 1.84 ±0.58 to4. 55 ± 0. 32 (P<0.01), MMF was significantly increased (P< 0.05) and the activities of FO F1 -ATPase and Na+ -K+ -ATPasewere increased by 66% and 25%, respectively. It is concluded that HP is efficacious to improve myocardial energy metabolismthrough the mechanism of the increase of mitochondrial membrane fluidity and the improvement of mitochondrial respiratory function.

Hypoxia preconditioning protects Ca^(2+)-ATPase activation of intestinal mucosal cells against R/I injury in a rat liver transplantation model

AIM To investigate the effect of ischaemia and reperfusion(I/R) injury on the Ca^(2+)-ATPase activation in the intestinal tissue of a rat autologous orthotopic liver transplantation model and to determine if hypoxia preconditioning(HP) therapy induces HIF-1α to protect rat intestinal tissue against I/R injury.METHODS Rats received non-lethal hypoxic preconditioning therapy to induce HIF-1α expression. We used an autologous orthotopic liver transplantation model to imitate the I/R injury in intestinal tissue. Then, we detected the microstructure changes in small intestinal tissues, Ca^(2+)-ATPase activity, apoptosis, and inflammation within 48 h postoperatively. RESULTS HIF-1α expression was significantly increased in intestinal tissue at 12 h postoperatively in rats that were exposed to a hypoxic environment for 90 min compared with a non-HP group(HP vs AT, P = 0.0177). Pathological analysis was performed on the intestinal mucosa cells, and the cells in the HP group appeared healthier than the cells in the AT group. The Ca^(2+)-ATPase activity in the small intestinal cells in the AT group was significantly lower after the operation, and the Ca^(2+)-ATPase activity in the HP group recovered faster than that in the AT group at 6 h postoperatively(HP vs AT, P = 0.0106). BCL-2 expression in the HP group was significantly higher than that in the AT group at 12 h postoperatively(HP vs AT P = 0.0010). The expression of the inflammatory factors NO, SOD, IL-6, and TNF-α was significantly lower in the HP group than in the AT group.CONCLUSION Hypoxia-induced HIF-1α could protect intestinal mucosal cells against mitochondrial damage after I/R injury. HP could improve hypoxia tolerance in small intestinal mucosal cells and increase Ca^(2+)-ATPase activity to reduce the apoptosis of and pathological damage to intestinal cells. HP could be a useful way to promote the earlier recovery of intestinal function after graft procedure.

miRNA-21-5p is an important contributor to the promotion of injured peripheral nerve regeneration using hypoxia-pretreated bone marrow-derived neural crest cells

Our previous study found that rat bone marrow–derived neural crest cells(acting as Schwann cell progenitors)have the potential to promote long-distance nerve repair.Cell-based therapy can enhance peripheral nerve repair and regeneration through paracrine bioactive factors and intercellular communication.Nevertheless,the complex contributions of various types of soluble cytokines and extracellular vesicle cargos to the secretome remain unclear.To investigate the role of the secretome and extracellular vesicles in repairing damaged peripheral nerves,we collected conditioned culture medium from hypoxia-pretreated neural crest cells,and found that it significantly promoted the repair of sensory neurons damaged by oxygen-glucose deprivation.The mRNA expression of trophic factors was highly expressed in hypoxia-pretreated neural crest cells.We performed RNA sequencing and bioinformatics analysis and found that miR-21-5p was enriched in hypoxia-pretreated extracellular vesicles of neural crest cells.Subsequently,to further clarify the role of hypoxia-pretreated neural crest cell extracellular vesicles rich in miR-21-5p in axonal growth and regeneration of sensory neurons,we used a microfluidic axonal dissociation model of sensory neurons in vitro,and found that hypoxia-pretreated neural crest cell extracellular vesicles promoted axonal growth and regeneration of sensory neurons,which was greatly dependent on loaded miR-21-5p.Finally,we constructed a miR-21-5p-loaded neural conduit to repair the sciatic nerve defect in rats and found that the motor and sensory functions of injured rat hind limb,as well as muscle tissue morphology of the hind limbs,were obviously restored.These findings suggest that hypoxia-pretreated neural crest extracellular vesicles are natural nanoparticles rich in miRNA-21-5p.miRNA-21-5p is one of the main contributors to promoting nerve regeneration by the neural crest cell secretome.This helps to explain the mechanism of action of the secretome and extracellular vesicles of neural crest cells in repairing damaged peripheral nerves,and also promotes the application of miR-21-5p in tissue engineering regeneration medicine.

P38MAPK磷酸化水平增高参与HPC降低MCAO所致小鼠缺血性脑损伤

目的探讨P38丝裂原激活蛋白激酶(P38 MAPK)磷酸化和蛋白表达水平在低氧预适应(HPC)降低脑中动脉阻塞(MCAO)所致缺血性脑损伤中的变化。方法利用已建小鼠HPC-MCAO模型,将健康雄性BALB/c小鼠随机分为常氧假手术(H0 Sham)、HPC假手术(H4 Sham)、常氧缺血(H0)和HPC缺血(H4)4组,应用2,3,5-氯化三苯基四氮唑(TTC)染色、N issl染色等方法观察脑损伤情况,应用W estern b lot并结合Gel Doc凝胶成像系统,定量检测小鼠脑组织内P38 MAPK磷酸化和蛋白表达水平的变化。结果HPC可明显减小MCAO所致的脑梗死体积(P<0.05)。与H0 sham相比,缺血组小鼠皮层缺血核心区和半影区P38 MAPK磷酸化水平显著升高(P<0.05,n=6),HPC可进一步增加缺血半影区和对侧皮层组织中P38 MAPK磷酸化水平(P<0.05,n=6)。各组间P38MAPK蛋白表达量水平无明显变化。结论P38 MAPK可能参与了HPC降低MCAO所致小鼠缺血性脑损伤的作用。

长链非编码核糖核酸肺腺癌转移相关转录本1和核旁斑组装转录本1在低氧预适应小鼠海马神经保护中的作用研究

目的探索长链非编码核糖核酸(lncRNA)肺腺癌转移相关转录本1(MALAT1)和核旁斑组装转录本1(NEAT1)在低氧预适应(HPC)小鼠海马细胞中的表达及其与神经保护的关系。方法(1)将36只雄性美国癌症研究所(ICR)小鼠按照随机数字表法完全随机分为3组:对照组、低氧组和低氧预处理组,每组12只。对照组小鼠不进行低氧暴露,低氧组小鼠低氧暴露1次,低氧预处理组小鼠低氧暴露4次。低氧处理结束后立即将所有小鼠脱颈椎处死并分离海马组织分组保存。(2)将HT22细胞培养于含10%胎牛血清和100 U/ml青霉素-链霉素的培养基,细胞汇合率大于90%时将其转移至24孔板中培养后分2批进行处理。通过转染试剂将6 pmol的乱序小干扰核糖核酸(siRNA)、MALAT1 siRNA(siMALAT1)、siNEAT1、siMALAT1+siNEAT1分别一一对应转染至第一批HT22细胞的阴性对照组、siMALAT1组、siNEAT1组、siMALAT1+siNEAT1组细胞中,空白组不做任何处理;然后在正常条件下(5%CO_(2)和95%空气)培养48 h;第二批HT22细胞中,利用转染试剂分别将6 pmol的乱序siRNA、乱序siRNA、siMALAT1、siMALAT1、siNEAT1、siNEAT1分别一一对应转染至阴性对照组、阴性对照+氧糖剥夺-再灌注(OGD/R)组、siMALAT1组、siMALAT1+OGD/R组、siNEAT1组、siNEAT1+OGD/R组的HT22细胞中,转染后48 h将阴性对照组、siMALAT1组、siNEAT1组HT22细胞在正常条件下(5%CO_(2)和95%空气)继续培养,将阴性对照+OGD/R组、siMALAT1+OGD/R组、siNEAT1+OGD/R组细胞进行OGD/R处理,即低氧条件下(1%O_(2)+5%CO_(2)+94%N_(2))暴露8 h,之后再进行正常条件下培养16 h。(3)通过实时荧光定量聚合酶链反应(PCR)及蛋白免疫印迹法测定各组小鼠海马组织中MALAT1、NEAT1、N-甲基-D-天冬氨酸受体亚基2B(NR2B)信使核糖核酸(mRNA)、NR2B蛋白水平的相对表达量和各组HT22细胞转染处理后NR2B mRNA、NR2B蛋白水平的相对表达量及各组HT22细胞转染和OGD/R后的血影蛋白分解产物、活化半胱氨酸蛋白酶蛋白3的相对表达量,并计算各组HT22细胞存活率。结果(1)3组小鼠海马中MALAT1(F=43.92)、NEAT1(F=506.40)、NR2B mRNA(F=50.64)及NR2B蛋白(F=41.24)的相对表达量差异均有统计学意义(均P<0.05)。与对照组相比,低氧组MALAT1[(1.68±0.06)比(1.00±0.08)]、NR2B mRNA[(1.26±0.06)比(1.00±0.01)]及NR2B蛋白[(1.47±0.05)比(1.00±0.01)]的相对表达量均增加(均P<0.05),而NEAT1[(1.02±0.10)比(1.00±0.03)]的相对表达量组间差异无统计学意义(P>0.05),低氧预处理组中MALAT1[(1.12±0.13)比(1.00±0.08)]和NEAT1[(2.88±0.10)比(1.00±0.03)]的相对表达量增加;与低氧组比较,低氧预处理组NR2B mRNA[(0.54±0.07)比(1.26±0.06)]及NR2B蛋白[(1.17±0.07)比(1.47±0.05)]的相对表达量均降低(均P<0.05)。(2)5组HT22细胞转染后NR2B mRNA(F=36.92)及NR2B蛋白(F=56.98)的相对表达量差异均有统计学意义(均P<0.05)。与阴性对照组相比,siMALAT1组[NR2B mRNA:(2.04±0.08)比(0.94±0.04),NR2B蛋白:(1.72±0.13)比(0.93±0.02)]、siNEAT1组[NR2B mRNA:(2.15±0.13)比(0.94±0.04),NR2B蛋白:(1.87±0.46)比(0.93±0.02)]、siMALAT1+siNEAT1组[NR2B mRNA:(2.09±0.16)比(0.94±0.04),NR2B蛋白:(2.07±0.30)比(0.93±0.02)]的NR2B mRNA及NR2B蛋白的相对表达量均增加(均P<0.05)。(3)6组HT22细胞转染及OGD/R处理后血影蛋白分解产物(145/150 kDa蛋白;F=12.43)、血影蛋白分解产物(120 kDa蛋白;F=7.15)、活化的半胱氨酸蛋白酶蛋白3蛋白(F=6.61)的相对表达量差异均有统计学意义(均P<0.05)。与siMALAT1组比较,siMALAT1+OGD/R组的145/150 kDa[(1.42±0.48)比(0.85±0.34)]、120 kDa[(1.33±0.37)比(0.52±0.19)]的血影蛋白分解产物及活化的半胱氨酸蛋白酶蛋白3蛋白[(2.43±0.35)比(1.15±0.24)]相对表达量均增加(均P<0.05);与阴性对照+OGD/R组比较,siMALAT1+OGD/R组的145/150 kDa[(1.42±0.48)比(1.23±0.17)]、120 kDa[(1.33±0.37)比(0.80±0.21)]的血影蛋白分解产物及活化的半胱氨酸蛋白酶蛋白3蛋白[(2.43±0.35)比(1.46±0.39)]相对表达量均增加(均P<0.05);与siNEAT1组比较,siNEAT1+OGD/R组的145/150 kDa[(1.28±0.44)比(0.87±0.32)]、120 kDa[(0.81±0.36)比(0.63±0.16)]的血影蛋白分解产物及活化的半胱氨酸蛋白酶蛋白3蛋白[(1.51±0.45)比(1.01±0.27)]相对表达量均增加(均P<0.05)。(4)6组HT22细胞存活率差异有统计学意义(F=5.54,P<0.05)。与阴性对照组比较,siMALAT1组、siNEAT1组、siMALAT1+OGD/R组及siNEAT1+OGD/R组HT22细胞存活率均降低[(0.65±0.40)、(0.76±0.35)、(0.24±0.17)、(0.23±0.16)比(0.84±0.04),均P<0.05];与siMALAT1组比较,siMALAT1+OGD/R组细胞存活率降低[(0.24±0.17)比(0.65±0.40),P<0.05];与siNEAT1组比较,siNEAT1+OGD/R组细胞存活率降低[(0.23±0.16)比(0.76±0.35),P<0.05]。结论HPC增加了小鼠海马组织中MALAT1和NEAT1的表达,MALAT1和NEAT1可能通过影响NR2B的表达参与小鼠缺血缺氧后的神经保护作用。

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。

The effect of ischemic precondition to IL-6 on rat liver ischemiareperfusion injury in transplantation

Objective:To investigate the effect of ischemic precondition to protect ischemia-reperfusion injury and reduce IL-6 expression in the rats liver transplantation.Methods:The rat portal vein infusion of autologous liver transplantation model were used.The rats were divided into ischemic preconditioning rats liver transplantation group(A group),the rats liver transplantation group(B group) and the normal rat control group(C group).Then we analyzed the changes of liver function,liver microstructure and the expression of IL-6,SOD and MDA within 48 h.Results: The pathology of liver in group A showed lobular architecture essentially normal,the liver cells was slightly swell and no significant changes in postoperative 12 h.In transmission electron microscope(46 000X).the mitochondria of liver cells in group A i】ecame swelling,elliptical can cristae partially broken.But there still has a small amount of arrangement.While that in group, the mitochondria were swollen,became round,serious visible crest reduce or ruptured.The result of over function test showed that the serum ALT and AST levels in group A and B were both higher than that in group C at each time period,but the serum ALT and AST levels in group A were lower than that in group B.The expression changes of IL-6 in group B were higher than that in group A and R(P【0.05).The expression of MDA in group A is more obvious than that in group B(P【0.05).Conclusions:Ischemic precondition could alleviate part of ischemia-reperfusion injury in the rat liver transplantation,and also could reduce IL-6 expression to protect the liver cells against liver damage and inflammatory cytokine production.

Priming of the Cells： Hypoxic Preconditioning for Stem Cell Therapy

Objective： Stem cell-based therapies are promising in regenerative medicine for protecting and repairing damaged brain tissues after injury or in the context of chronic diseases. Hypoxia can induce physiological and pathological responses. A hypoxic insult might act as a double-edged sword, it induces cell death and brain damage, but on the other hand, sublethal hypoxia can trigger an adaptation response called hypoxic preconditioning or hypoxic tolerance that is of immense importance for the survival of cells and tissues. Data Sources： This review was based on articles published in PubMed databases up to August 16, 2017, with the following keywords：＂stem cells,＂ ＂hypoxic preconditioning,＂ ＂ischemic preconditioning,＂ and ＂cell transplantation.＂Study Selection： Original articles and critical reviews on the topics were selected. Results： Hypoxic preconditioning has been investigated as a primary endogenous protective mechanism and possible treatment against ischemic injuries. Many cellular and molecular mechanisms underlying the protective effects of hypoxic preconditioning have been identified. Conclusions： In cell transplantation therapy, hypoxic pretreatment of stem cells and neural progenitors markedly increases the survival and regenerative capabilities of these cells in the host environment, leading to enhanced therapeutic effects in various disease models. Regenerative treatments can mobilize endogenous stem cells for neurogenesis and angiogenesis in the adult brain. Furthermore, transplantation of stem cells/neural progenitors achieves therapeutic benefits via cell replacement and/or increased trophic support. Combinatorial approaches of cell-based therapy with additional strategies such as neuroprotective protocols, anti-inflammatory treatment, and rehabilitation therapy can significantly improve therapeutic benefits. In this review, we will discuss the recent progress regarding cell types and applications in regenerative medicine as well as future applications.

Hypoxic preconditioning in an autohypoxic animal model

Hypoxic preconditioning refers to the exposure of organisms, systems, organs, tissues or cells to moderate hypoxia/ischemia that results in increased resistance to a subsequent episode of severe hypoxia/ischemia. In this article, we review recent research based on a mouse model of repeated exposure to autohypoxia. Pre-exposure markedly increases the tolerance to or protection against hypoxic insult, and preserves the cellular structure of the brain. Furthermore, the hippocampal activity amplitude and frequency of electroencephalogram, latency of cortical somatosensory-evoked potential and spinal somatosensory-evoked potential progressively decrease, while spatial learning and memory improve. In the brain, detrimental neurochemicals such as free radicals are down-regulated, while beneficial ones such as adenosine are up-regulated. Also, antihypoxia factor（s） and gene（s） are activated. We propose that the tolerance and protective effects depend on energy conservation and plasticity triggered by exposure to hypoxia via oxygen-sensing transduction pathways and hypoxia-inducible factor-initiated cascades. A potential path for further research is the development of devices and pharma-ceuticals acting on antihypoxia factor（s） and gene（s） for the prevention and treatment of hypoxia and related syndromes.