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BACKGROUND: Previous study demonstrated that hypoxia preconditioning promoted mesenchymal stem cells survival and their therapeutic efficacy, and this effect was mediated by hypoxia induced factor-1α (HIF-1α). However, specific downstream mechanism remained unclear. OBJECTIVE: To observe the influence of hypoxia preconditioning on the survival and vascularization potential of bone marrow mesenchymal stem cells in vitro and explore the regulatory mechanism of HIF-1α/MALAT1/VEGFA pathway. METHODS: Bone marrow mesenchymal stem cells were obtained and cultured in vitro. Cells were divided into hypoxia (1% O2) and normoxia control groups (20% O2), and cultured for 24 hours. Cells proliferation, apoptosis and vascularization were evaluated. The expression of HIF-1α, MALAT1, and VEGFA was detected. HIF-1α and MALAT1 were inhibited by their siRNAs separately. HIF-1α siRNA scramble and MALAT1 siRNA scramble were used as negative controls before hypoxia preconditioning. Alterations of the molecules were examined and compared in different groups. RESULTS AND CONCLUSION: (1) Compared with the normoxia control group, cell viability was significantly enhanced; and cell apoptosis percentage was significantly declined in the hypoxia group; vascular lumen like structure was also increased significantly in the hypoxia group (P < 0.01); expression of HIF-1α, MALAT1, and VEGFA was significantly increased in the hypoxia group (P < 0.01). (2) After the inhibition of HIF-1α and hypoxia preconditioning, both MALAT1 and VEGFA expression levels were significantly reduced (P < 0.01). The expression of VEGFA was also significantly suppressed after the blockage of MALAT1 (P < 0.01). (3) This study suggested that hypoxia preconditioning effectively promoted bone marrow mesenchymal stem cell survival and vascularization through the activation of HIF-1α/MALAT1/VEGFA pathway.
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Although previous and ongoing clinical studies have used stromal cells during renal ischemia-reperfusion injury (IRI), there is little consensus regarding the optimal protocol. We aimed to optimize the protocol for hypoxic preconditioned human bone marrow-derived mesenchymal stromal cell (HP-hBMSC) therapy in a rat model of renal IRI. We determined the optimal injection route (renal arterial, renal parenchymal, and tail venous injection), dose (low-dose: 1×10⁶, moderate-dose: 2×10⁶, and high-dose: 4×10⁶), and injection period (pre-, concurrent-, and post-IRI). During optimal injection route study, renal arterial injections significantly reduced the decreasing glomerular filtration rate (GFR), as compared to GFRs for the IRI control group, 2 and 4 days after IRI. Therapeutic effects and histological recoveries were the greatest in the group receiving renal arterial injections. During the dose finding study, high-dose injections significantly reduced the decreasing GFR, as compared to GFRs for the IRI control group, 3 days after IRI. Therapeutic effects and histological recoveries were the greatest in the high-dose injection group. While determining the optimal injection timing study, concurrent-IRI injection reduced elevated serum creatinine levels, as compared to those of the IRI control group, 1 day after IRI. Pre-IRI injection significantly reduced the decreasing GFR, as compared with GFRs for the IRI control group, 1 day after IRI. Therapeutic effects and histological recoveries were the greatest in the concurrent-IRI group. In conclusion, the concurrent-IRI administration of a high dose of HP-hBMSC via the renal artery leads to an optimal recovery of renal function after renal IRI.
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Animals , Humans , Rats , Acute Kidney Injury , Cell- and Tissue-Based Therapy , Consensus , Creatinine , Glomerular Filtration Rate , Mesenchymal Stem Cells , Models, Animal , Renal Artery , Reperfusion Injury , Stromal Cells , Tail , Therapeutic UsesABSTRACT
Objective To preliminarily explore the effects and brain protective mechanism of intermittent hypoxia preconditioning ( IHP) on rats with seizures induced by lithium-pilocarpine ( Li-pilo) .Methods A total of 96 8-week old male Sprague Dawley rats ( clean grade ) were randomly divided into control group , seizure group and four IHP-seizure groups.The animal model of epilepsy was established by intraperitoneal injection of Li-pilo in the seizure group and four IHP-seizure groups (Li-pilo was injected at 1, 3, 7, or 14 days after a 5-day regimen of IHP).Subsequent seizure behavior , the latency period and percentage of generalized seizures were quantitatively evaluated for 240 min and the cognitive function was tested by Morris water maze task , and followed by the detection of hippocampus neuron apoptosis and related protein (BCL-2, Bax, and cleaved-caspase-3) by TUNEL labeling and Western blot, respectively.Results The induced seizure peaked on an average between 50-150 min after Li-pilo administration , scored using a modified Racine scale.The average scores of modified Racine scale in the IHP-3d seizure group was significantly lower than that in the other groups.The latency period and percentage of generalized seizures in the IHP-3d seizure group rats were significantly different from the parameters in the seizure group rats (P0.05).Conclusions The results indicate that IHP treatment may help to decrease the susceptibility to epilepsy by reducing abnormal apoptosis , and has a brain protective effect on the seizure rats .
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Aim To screen the differentially expressed microRNAs ( miRNAs ) induced by hypoxia precondi-tioning ( HPC ) in adult rat cardiomyocytes, and pre-dict miRNAs-regulated target genes and their func-tions. Methods Cardiomyocytes were isolated from a-dult rat ventricular myocardium and cultured ( in vitro) . The cells were divided into 2 groups: control group ( CON ) and hypoxia preconditioning group ( HPC) . The cardiomyocytes in HPC group were sub-jected to 10 min hypoxia followed by 30 min reoxygen-ation, while the cells in CON group were cultured un-der normal condition. After that, total RNA was ex-tracted and then subjected to miRNA microarray to screen differentially expressed miRNAs. The microar-ray results were further validated by quantitative RT-PCR ( qRT-PCR ) . Bioinformatics analysis was per-formed to predict the miRNAs-regulated target genes and analyze the enriched gene ontology ( GO) and sig-naling pathway ( Pathway) . Results HPC caused sig-nificant changes in miRNAs expression in cardiomyo-cytes as compared to CON group. A total of 12 miR-NAs were up-regulated and 14 miRNAs were down-reg-ulated ( P 500 were selected for further bioinformatics analysis. The expression of miR-133b-5p, miR-664-1-5p and miR-6216 detected by qRT-PCR exhibited the similar patterns of up or down regulation to those shown in mi-croarray results. Bioinformatics analysis revealed that miRNAs-regulated target genes were significantly en-riched in 27 GOs and 6 signal pathways. Conclusion The expression profile of miRNAs in rat cardiomyo-cytes is significantly affected by HPC. These differenti-ally expressed miRNAs might participate in HPC-in-duced cardioprotection by regulating their target genes in rat cardiomyocytes.
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Objective To observe the effects of intermittent hypoxic preconditioning on the expression of apoptosis-related EPO after 70％ hepatectomy combined with ischemia-reperfusion injury.Methods One hundred and twenty healthy SD rats of clean grade,simple random divided into 3 groups:Sham operation group of 40 rats ; Pure major hepatic resection group was 40 (Major hepatectomy,MH),namely in the hepatic portal blocking liver resection of the left and middle lobes,hepatic portal blocking 20 min ; intermittent hypoxia preconditioning group 40 (Intermittent hypoxia preconditioning,IHP group).Results MH group,S group,IHP group,EPO level in three experimental groups in postoperative residual liver tissue in rats with significant difference (P ＜ 0.05),Intermittent hypoxia preconditioning group of residual liver EPO content was significantly higher than that of simple hepatic resection group 40.Conclusion Intermittent hypoxia preconditioning can promote the expression of residual liver after hepatectomy in EPO.
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Objective To observe the effects of intermittent hypoxic preconditioning on the expression of apoptosis-related Bcl-2 and Bax protein after 70％ hepatectomy combined with ischemia-reperfusion injury.Methods A total of fifty-four SD rats were randomly divided into three groups (n =18).Partial hepatectomy hroup (PH Group):Rats underwent the left and middle lobectomy of liver(70％ hepatectomy).Ischemia reperfusion group (IR group):The left and middle lobes of liver were resected during the occlusion of the hepatoduodenal ligament for 20 minutes.Residual liver underwent the process of ischemia-reperfusion.Intermittent hypoxia preconditioning group (IHP group):rats were exposed to hypoxic environment of 10％ oxygen for 1 h/d.After 7 consecutive days,the left and middle lobes of liver were resected under the portal triad clamping.At 12,24 and 48 hours after the operation,the rats were killed and detected.The serum levels of ALT and AST were determined by automatic biochemical analyzer.The expression of Bcl-2 and Bax protein in liver tissue were measured by immunohistochemistry.Results At each time point after surgery,the serum levels of ALT and AST in IR group and IHP group were higher than that of PH group,and IHP group were lower than in IR group.Compared with IR group,the expression of Bcl2 protein significantly increased and Bax protein expression significantly decreased in IHP group.All these differences were statistically significant (P ＜ 0.05).Conclusions Intermittent hypoxic preconditioning might protect residual liver against ischemia reperfusion injury,through increasing the expression of Bcl-2 protein and reducing the expression of Bcl-2 protein to decrease liver cell apoptosis.
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Recent accumulating studies have reported that hypoxic preconditioning during ex vivo expansion enhanced the self-renewal or differentiation of various stem cells and provide an important strategy for the adequate modulation of oxygen in culture conditions, which might increase the functional bioactivity of these cells for cardiac regeneration. In this study, we proposed a novel priming protocol to increase the functional bioactivity of cardiac progenitor cells (CPCs) for the treatment of cardiac regeneration. Firstly, patient-derived c-kit+ CPCs isolated from the atrium of human hearts by enzymatic digestion and secondly, pivotal target molecules identified their differentiation into specific cell lineages. We observed that hCPCs, in response to hypoxia, strongly activated ERK phosphorylation in ex vivo culture conditioning. Interestingly, pre-treatment with an ERK inhibitor, U0126, significantly enhanced cellular proliferation and tubular formation capacities of CPCs. Furthermore, we observed that hCPCs efficiently maintained the expression of the c-kit, a typical stem cell marker of CPCs, under both hypoxic conditioning and ERK inhibition. We also show that hCPCs, after preconditioning of both hypoxic and ERK inhibition, are capable of differentiating into smooth muscle cells (SMCs) and cardiomyocytes (CMs), but not endothelial cells (ECs), as demonstrated by the strong expression of alpha-SMA, Nkx2.5, and cTnT, respectively. From our results, we conclude that the functional bioactivity of patient-derived hCPCs and their ability to differentiate into SMCs and CMs can be efficiently increased under specifically defined culture conditions such as short-term hypoxic preconditioning and ERK inhibition.
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Humans , Hypoxia , Cell Lineage , Cell Proliferation , Digestion , Endothelial Cells , Healthcare Common Procedure Coding System , Heart , Myocytes, Cardiac , Myocytes, Smooth Muscle , Oxygen , Phosphorylation , Regeneration , Stem CellsABSTRACT
Objective Identify novel protein kinase Cε(nPKCε)-interacted proteins in the cortex of hypoxic preconditioned mice.Methods Immunoprecipitation (IP) and two-dimensional electrophoresis (2-DE) combining with ImageMaster 2D Platinum software were applied to analyze the differential expressions of nPKCe-interacted proteins;the target protein spots were identified by matrix-associated laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and Western blot.Results Compared with control group,there were 34 upregulated protein spots and 20 downregulated protein spots in cytosolic fraction,while 27 upregulated prtein spots and 28 downregulated protein spots were determined in particulate fraction of cerebral cortex of HPC mice.The levels of nPKCε-interacted HSP 70 and 14-3-3γ/protein expressions increased significantly in both cytosolic and particulate fractions;but the protein level of nPKCε-interacted HSP60 increased only in particulate fraction of cerebral cortex of HPC mice.Conclusion nPKCε might be involved in the development of cerebral HPC via the regulation of its interacted proteins such as HSP60,HSP70 and 14-3-3γ.
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Objective To compare the protecting effect of hypoxia preconditioning(HP) and deferoxam-ine preconditioning (DP)on transplanted livers and to search a protecting method which is suitable for clini-cal practice.Methods SD rats are randomly divided into four groups:sham operation group (S),analogic transplantation group (AT),deferoxamine preconditioning group (DP) and hypoxia preconditioning group (HP).Twenty-four hours after operation,several factors of each group were measured respectively,inclu-ding deterioration of histomorphology,hepatic zymogram in the serum,hypoxia inducing factor-1 (HIF-1α)and malonaldehyde (MDA) in hepatic tissue.Results The levels of alanine aminotransferase (ALT),glu-tamic oxalacetic transaminase (AST) and alkaline phosphatase (ALP) in AT group,DP group and HP group were higher than S group's.Levels of ALT,AST,ALP in AT group were dramatically high,hepatic tissue suffers from edema,degeneration even necrosis.By contrast,the pathological change is milder in DP group and HP group.HIF-1α in hepatic tissue of AT group was lower than that in DP group and HP group,MDA was higher than two latters (P < 0.05).Conclusion DP can protect transplanted liver as same as HP,however,DP is more suitable for clinical practice.
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@#Brain ischemia/hypoxia preconditioning(I/HPC) is one kind of endocardial protective phenomenon by organism against blood and oxygen deficit.Researches discovered various proteins such as hypoxic inducible factor-1(HIF-1),heat shock proteins(HSPs),erythropoietin(EPO),Bcl-2 family,calcitoningene related peptide(CGRP) and so on participated in brain I/HPC.This essay explains function and participating I/HPC mechanism of these proteins.
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@#Brain ischemia/hypoxia preconditioning(I/HPC) is one kind of endocardial protective phenomenon by organism against blood and oxygen deficit.Researches discovered various proteins such as hypoxic inducible factor-1(HIF-1),heat shock proteins(HSPs),erythropoietin(EPO),Bcl-2 family,calcitoningene related peptide(CGRP) and so on participated in brain I/HPC.This essay explains function and participating I/HPC mechanism of these proteins.
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Objective To observe the effects of hypoxia preconditioning on the mitochondria ultrastructure of hepatocyte during liver transplantation in rats.Methods A modified orthopotic liver autotransplantation model was used to simulate liver transplantatation.Seventy two Sprague-Dawley rats were randomly divided into the following three groups:the normal control(group NC),the autotransplantation group(group AT),and the hypoxia preconditioning group(group HP),with twenty four rats in each group.Group HP were given an 8% oxygen atmosphere for 90 minutes before the operation.At 1,6,and 24 hours after the operation,the rats were killed and the following tests were conducted:1.The morphology of mitochondria was observed under transmission electron microscopy(TEM);2.Mitochondria were quantitatively analyzed by a MiVnt image analysis system.ResultsHepatic cells in group AT showed typical injury signs under TEM,the appearance of hepatocytes and mitochondria in group HP were much better than the group AT,and the areas,perimeters and diameters of mitochondria in group HP were much smaller than those in group AT by a significantly amount(P
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Objective To investigate the roles of hypoxia regulated genes/protein in chemical hypoxia preconditioning in differentiated SH-SY5Y cells.Methods Differentiated SH-SY5Y cells were randomly divided into control group,chemical hypoxic preconditioning group(50 ?mol/L CoCl2 preconditioning for 3 h,normal culture for 1 h,then in 2%O2 for 28 h) and hypoxia group(in 2%O2 for 28 h).RT-PCR was used to examine the mRNA expressions of VEGF,GLUT-1,EPO,LDH-A.Further evaluation of the potential neuroprotective effect of VEGF was done by investigating the effect of recombinant human VEGF on subsequent hypoxia injury.Results The mRNA levels of GLUT-1,EPO increased in the preconditioning group as compared with those in the hypoxia group(P