Effects of 24-Epibrassinolide on Antioxidant System in Cucumber Seedling Roots Under Hypoxia Stress

This article aims to study the effects of exogenous 24-epibrassinolide （EBR） on the changes in ROS, activities of antioxidative enzymes and antioxidants in cucumber （Cucumis sativus L.） seedling roots under hypoxia stress. Seedlings of a hypoxiaresistant cultivar, Lühachun 4, and a hypoxia-sensitive cultivar, Zhongnong 8, were hydroponically grown for 8 d in normoxic or hypoxic nutrient solutions that were added or not added with 10^-3 mg L^-1 EBR. Under hypoxia stress, the ROS levels and the lipid peroxidation were significantly increased in the roots upon exposure to hypoxia stress, which were inhibited by EBR application. The EBR treatment significantly increased the seedlings growth and SOD, APX, GR activities, and contents of AsA and GSH under hypoxia stress. From the results obtained in this study, it can be concluded that oxidative damage on seedling roots by hypoxia stress can be considerably alleviated and the tolerance of plants was elevated.

Resistant Evaluation of Kiwifruit Rootstocks to Root Zone Hypoxia Stress

In this thesis, 10 species of kiwifruit rootstocks were treated with hydroponics hypoxia to study their root zone hypoxia tolerance. The results were as follows: growth of all kiwifruit seedlings was inhibited. The max length of new root, plant height, plant biomass, root activity, relative growth rate of leaves, and content of chlorophyll in leaves under root zone hypoxia stress obviously declined comparing with control. MDA content, relative conductance in the leaves and roots all increased in 10 kinds of kiwifruit seedlings. The sensitivities of 10 kinds’ kiwifruit seedlings to hypoxia stress were obviously different. With the method of subordinate function and cluster analysis, the adversity resistance coefficient of 10 kinds’ kiwifruit seedlings, were comprehensively evaluated in order to appraise their hypoxia-tolerance abilities. According to the results, “Hayward”, “Qinmei”, “Jinxiang”, “Kuoye”, “Huayou” kiwifruit seedlings held higher tolerance to root zone hypoxia stress, while “Hongyang” kiwifruit seedlings were sensitive to root zone hypoxia stress. The others, including “Xixuan”, “Maohua”, “Jinhua”, “Shanli” kiwifruit seedlings kept moderate resistant intensity to root zone hypoxia stress. The kiwifruit seedlings’ resistance order from strong to weak was: “Hayward” > “Qinmei” > “Jinxiang” > “Kuoye” > “Huayou” > “Xixuan” > “Maohua” > “Jinhua” > “Shanli” > “Hongyang”.

Effects of Hypoxia Stress and Different Level of Mn2+ on Antioxidant Enzyme of Tomato Seedlings

The changes of antioxidant enzyme activities and related genes expression of tomato seedlings were evaluated under hypoxia stress with different levels of Mn2+. Activities of superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxide (APX), glutathione reductase (GR), catalase (CAT), the contents of H2O2, ascorbic (AsA) and malondialdehyde (MDA) were studied to investigate how active oxygen damaged the membrane lipid under hypoxia stress. With 10-200 μmol?L-1 Mn2+, the activities of SOD, POD, APX, GR and the contents of H2O2, AsA, MDA of leaves and roots increased significantly, which indicated that low Mn2+ could eliminate the active oxygen and protect the membrane lipid from hurt. But the activities of catalase (CAT) decreased evidently in the root. When the concentration of Mn2+ reached 400-600 μmol?L-1 under hypoxia stress, the activities of SOD, POD, APX, GR and ASA content decreased remarkably. However, the contents of H2O2 and MDA increased contrarily. A series of resistance genes level achieved peak value with 10 μmol?L-1 Mn2+. The expression level of SOD, CAT, APX, POD, GR were 6.28, 2.19, 5.66, 5.21 and 6.79 times compared to control respectively. These results illustrated appropriate amount of Mn2+ could reduce the damage of active oxygen under hypoxia stress, but reversely, high level of Mn2+ just aggravated the already serious damage to the tomato seedlings.

Effects of Intermittent Hypoxia Exposure on Symptoms of Chronic Fatigue Syndrome in Repeated Restraint Stress and Forced Swimming Induced-Mouse Model

Background: Chronic fatigue syndrome (CFS) shows as its main symptoms debilitating fatigue that is not relieved by physiological rest, depression, inflammation, learning disability and memory impairment. But, intermittent hypoxia, consisting of alternating exposure to hypoxia and normoxia, plays a very important role in improving CFS. However, the essential components for improving learning and memory in CFS patients as well as their mechanism are largely unknown. Objectives: This study aims to analyze the effects of 12% and 15% hypoxia on the expression of alpha tumor necrosis factor (TNF-α) and nuclear factor kappa B (NF-κB) in CFS induced-mouse model for clarifying the effects on the learning and memory function. Methods: A total of 48 type IC mice were used. The CFS mouse model was established using restrained stress and repeated forced swimming. Treatment of CFS was done by exposing CFS mice to intermittent hypoxia at 12% and 15%. The effects of intermittent hypoxia on learning and memory as well as its mechanism of action on inflammation were tested respectively with the Morris test, the SDS page, the immunohistochemistry technique and the Nissl staining. Results: We found that 12% and 15% intermittent hypoxia exposure improved learning capacity and memory of CFS induced-mice. SDS page showed that CFS caused higher TNF-α expression. By exposing CFS mice to 12% and 15% intermittent hypoxia, TNF-α expression decreased significantly, with a much better effect at 15%. Both TNF-α and NF-κB increased in CFS state and decreased after treatment with intermittent hypoxia. Conclusion: Intermittent hypoxia improves learning capacity and memory. It acted by decreasing NF-κB come to down-regulating TNF-α and ameliorates learning capacity and memory impairment in CFS mice.

Vital contribution of brassinosteroids to hypoxia-stimulated coleoptile elongation in submerged rice

The rapid elongation of rice(Oryza sativa)coleoptile is pivotal for the plant plumule to evade hypoxia stress induced by submergence,a condition often arising from overirrigation,ponding,rainstorms,or flooding.While brassinosteroids(BRs)are recognized for their diverse roles in plant growth and development,their influence on coleoptile elongation under hypoxic conditions remains largely unexplored.In this study,we demonstrate the significant requirement of BRs for coleoptile elongation in deep water.During coleoptile development,Glycogen Synthase Kinase3-Like Kinase2(GSK2),the central inhibitor of BR signaling in rice,undergoes substantial suppression in deep water but induction in air.In contrast,the dephosphorylated form of BRASSINAZOLE RESISTANT1(OsBZR1),representing the active form of the key BR signaling transcription factor,is induced in water but suppressed in air.Remarkably,the knockout of GSK3-like kinase genes significantly enhances coleoptile elongation in deep water,strongly indicating a vital contribution of BR response to hypoxia-stimulated coleoptile elongation.Transcriptome analysis uncovers both BR-associated and BR-independent hypoxia responses,implicating substance metabolism,redox reactions,abiotic stress responses,and crosstalk with other hormones in the regulation of BR-induced hypoxia responses.In summary,our findings suggest that rice plumules rapidly elongate coleoptiles through the activation of BR response in deep water,enabling them to escape from submergence-induced hypoxia stress.

Effects of Hypoxia on Oxidative Stress, Autophagy and Apoptosis in Cardiomyocytes

Coronary heart disease (CHD) is a hypoxia related disease. However, the relationship of the hypoxia-induced oxidative stress, autophagy and apoptosis in cardiomyocyte remains unclear. In this study, we used CoCl2 to mimic hypoxic conditions in H9c2 cardiomyocytes and study the effects of CoCl2-induced hypoxia on oxidative stress, apoptosis and autophagy, as well as the relationships among these processes. Cell viability and levels of ROS, LC3-II, p62, caspase-3 and PARP were assessed. The viability and morphology of cardiomyocytes were affected by hypoxia, and hypoxia enhanced levels of ROS and the levels of the LC3-II, p62, caspase-3 and PARP proteins in H9c2 cells in a dose-dependent manner. ROS levels rise gradually in the presence of hypoxia;however, it shrinks when hypoxia reaches a certain level. Caspase-3 and PARP levels were raised with the increasing of hypoxia level. Enhanced level of LC3 and decreased levels of p62 in hypoxic cells indicate that autophagy levels are in accord with hypoxia. Based on these results, hypoxia induces oxidative stress, apoptosis and autophagy in cardiomyocytes. Autophagy is a double-edged sword. At a low level, autophagy can resist oxidative stress and protect cardiomyocytes from oxidative stress, while high level autophagy can promote apoptosis of cardiomyocytes.

Morphological and migratory alterations in retinal Müller cells during early stages of hypoxia and oxidative stress

In the present study, retinal MOiler cells were cultured in vitro and treated with hydrogen peroxide （oxidative stressor） and cobalt chloride （hypoxic injury）. Following 24 hours of culture, compensatory hypertrophy was observed and cellular apoptosis increased. Hypoxia enhanced the migration ability of retinal MOiler cells and induced the expression of a-smooth muscle actin. Oxidative stress altered the morphology of MOiler cells when compared with hypoxia treatment.

The ROCK pathway inhibitor Y-27632 mitigates hypoxia and oxidative stress-induced injury to retinal Müller cells

Rho kinase （ROCK） was the first downstream Rho effector found to mediate RhoA-induced actin cytoskeletal changes through effects on myosin light chain phosphorylation. There is abundant evidence that the ROCK pathway participates in the pathogenesis of retinal endothelial injury and proliferative epiretinal membrane traction. In this study, we investigated the effect of the ROCK pathway inhibitor Y-27632 on retinal Müller cells subjected to hypoxia or oxidative stress. Müller cells were subjected to hypoxia or oxidative stress by exposure to CoCl2 or H2O2. After a 24-hour treatment with Y-27632, the 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl tetrazolium bromide assay was used to assess the survival of Müller cells. Hoechst 33258 was used to detect apoptosis, while 2′,7′-dichlorodihydrofluorescein diacetate was used to measure reactive oxygen species generation. A transwell chamber system was used to examine the migration ability of Müller cells. Western blot assay was used to detect the expression levels of α-smooth muscle actin, glutamine synthetase and vimentin. After treatment with Y-27632, Müller cells subjected to hypoxia or oxidative stress exhibited a morphology similar to control cells. Y-27632 reduced apoptosis, α-smooth muscle actin expression and reactive oxygen species generation under oxidative stress, and it reduced cell migration under hypoxia. Y-27632 also upregulated glutamine synthetase expression under hypoxia but did not impact vimentin expression. These findings suggest that Y-27632 protects Müller cells against cellular injury caused by oxidative stress and hypoxia by inhibiting the ROCK pathway.

Adiponectin Attenuates Hypoxia/Reoxygenation-Induced Cardiomyocyte Injury Through Inhibition of Endoplasmic Reticulum Stress

Background and Aim Adiponectin(APN) is a potent cardioprotective molecule.The present study aims to investigate the under-lying mechanism(s) for its cardioprotective effect.Methods Primary cardiomyocytes were isolated from neonatal rats and an invitro model of hypoxia-reoxygenation(H/R) was established.The cardiomyocytes were randomly divided into six groups: salinegroup(control),dithiothreitol(DTT) group(5 mmol/L DTTfor 2 h),H/R group,H/R +APN group(incubation with 30 mg/LAPN,followed by H/R),H/R +APN +SB203580(SB) group(treatment with 30 mg/L APN and 5μmol/L SB,followed by H/R),and H/R +SB group(exposure to 5μmol/L SB and then H/R).Cell death was detected by measuring lactate dehydrogenase(LDH) release.The expression levels of hypoxia-inducible factor-1alpha(HIF-1α) and endoplasmic reticulum(ER) stress-relatedgenes including GRP78,caspase-12,C/EBP homologus protein(CHOP),and p38 mitogen-activated protein kinase(MAPK) wereexamined.Results Cardiomyocytes exposed to H/R showed a significant increase in LDH leakage and HIF-1αprotein levelscompared with the control cells(P<0.05).The H/R-provoked cell death was profoundly attenuated by the pretreatment with APNalone,SB alone,or both,which was coupled with decreased expression of GRP78,caspase-12,CHOP,and p38 MAPK.Conclu-sions These results provide new insights into the mechanism of APN-mediated cardioprotection,which may be partially due to inhibi-tion of ER stress response.

Regulation of heme oxygenase expression by alcohol,hypoxia and oxidative stress

AIM:To study the effect of both acute and chronic alcohol exposure on heme oxygenases(HOs) in the brain,liver and duodenum.METHODS:Wild-type C57BL/6 mice,heterozygous Sod2 knockout mice,which exhibit attenuated manganese superoxide dismutase activity,and liver-specific ARNT knockout mice were used to investigate the role of alcohol-induced oxidative stress and hypoxia.For acute alcohol exposure,ethanol was administered in the drinking water for 1 wk.Mice were pair-fed with regular or ethanol-containing Lieber De Carli liquid diets for 4 wk for chronic alcohol studies.HO expression was analyzed by real-time quantitative polymerase chain reaction and Western blotting.RESULTS:Chronic alcohol exposure downregulated HO-1 expression in the brain but upregulated it in the duodenum of wild-type mice.It did not alter liver HO-1 expression,nor HO-2 expression in the brain,liver or duodenum.In contrast,acute alcohol exposure decreased both liver HO-1 and HO-2 expression,and HO-2 expression in the duodenum of wild-type mice.The decrease in liver HO-1 expression was abolished in ARNT+/-mice.Sod2+/-mice with acute alcohol exposure did not exhibit any changes in liver HO-1 and HO-2 expression or in brain HO-2 expression.However,alcohol inhibited brain HO-1 and duodenal HO-2 but increased duodenal HO-1 expression in Sod2+/-mice.Collectively,these findings indicate that acute and chronic alcohol exposure regulates HO expression in a tissue-specific manner.Chronic alcohol exposure alters brain and duodenal,but not liver HO expression.However,acute alcohol exposure inhibits liver HO-1 and HO-2,and also duodenal HO-2 expression.CONCLUSION:The inhibition of liver HO expression by acute alcohol-induced hypoxia may play a role in the early phases of alcoholic liver disease progression.

Cloning of hemoglobin-α1 from half-smooth tongue sole (Cynoglossus semilaevis) and its expression under short-term hypoxia

This study cloned the hemoglobin a1 from the marine teleost, the half-smooth tongue sole （Cynoglossus semilaevis）, and then examined its expression under hypoxia exposure. The full-length of CsHb-a1 （594 bp） cDNA contains an open reading frame encoding 144 amino acids. Sequence analysis shows that the predicted CsHb-a1 amino acids shares high identities with that of other species. Real-time PCR showed that CsHb-a1 was highly expressed in the heart, liver, spleen, kidney and blood. Five to 120 min esposure and long-term （36 h） exposure to hypoxia （1.0 mg/L） significantly increased CsHb-a1 mRNA expression in most tissues compared to those fish held in normoxic conditions （dissolved oxygen （DO）： 6.2 mg/L）. These results suggested that the up-regulation of Hb-a1 is an important component for adaptation of half-smooth tongue sole to short-term hypoxia.

The timing and extent of intraosseous hypoxia in the oxidative stress-induced rat osteonecrosis model

Using a rat oxidative stress-induced femoral head osteonecrosis model, we determined the presence/ absence and timing of the generation of hypoxia in the femoral head. DL-Buthionine-(S,R)-sulfoximine (BSO) 500 mg/kg was administered intraperitoneally to male Wistar rats. The rats were killed at 1, 3, 6, 12 hours, and 1, 3, 5 days after BSO administration, and the bilateral femora were removed. A group not administered BSO (control group) was also studied (each group n = 5). In the femoral heads of each group, the expression of hypoxia-inducible factor-1 alpha (HIF-1α) as an index of hypoxia was confirmed by the Western blot method, and quantified using analytical software. In the femoral head increased HIF-1α expression was found in all groups from 1 hour after BSO administration (p < 0.05). In particular, in all specimens of the group 3 hours after BSO administration the most intense expression of HIF-1α amounting to about 13-fold of that of control group was noted (p < 0.001). The present results suggested that in the extremely short period of 3 hours after BSO administration hypoxia severe enough to cause osteonecrosis was induced by oxidative stress in the rat femoral head.

复方人参对机体缺氧损伤的保护作用及其分子机制研究

探究复方人参(compound ginseng,CGS)的抗缺氧作用及其分子机制。构建小鼠密闭缺氧模型、亚硝酸钠中毒模型、急性脑缺血模型以及全心缺血缺氧模型,观察CGS对小鼠的存活时间、喘气时间、喘气次数、丙二醛(malondialdehyde,MDA)含量、超氧化物歧化酶(superoxide dismutase,SOD)活力、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)活性、乳酸脱氢酶(lactic dehydrogenase,LDH)活力和肌酸激酶(creatine kinase,CK)活力。利用网络药理学预测CGS的抗缺氧相关机制,并采用分子对接方法验证网络药理学筛选所得CGS活性成分与机体靶点之间的亲和性,检测小鼠体内缺氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)和血管内皮生长因子(vascular endothelial growth factor,VEGF)mRNA表达量。结果显示,CGS可以显著延长密闭缺氧实验、亚硝酸钠中毒实验和全心缺血缺氧实验小鼠的存活时间以及急性脑缺血实验的喘气时间和喘气次数;CGS能够降低小鼠血清中的MDA含量、LDH和CK活力,提升SOD、GSH-Px活性。采用网络药理学预测出226条CGS抗缺氧通路,其中关键通路有HIF-1α通路;密闭缺氧实验结果证实CGS可以显著提升HIF-1α表达,下调VEGF表达。综上,CGS具有耐缺氧作用,其作用可能与激活HIF-1α/VEGF通路,提高血液携氧能力和氧利用率,增强抗氧化活性有关。

Comparative Transcriptome Analysis of Heart Tissue in Response to Hypoxia in Silver Sillago (Sillago sihama)

Sillago sihama,commonly known as silver sillago,is considered as an economically important fish species in China.It is sensitive to hypoxia stress in the larval stage,and the mechanism has not been understood thoroughly.In this study,we investigated the transcriptome change in heart tissues under hypoxia stress.The fish were divided into four groups,including 1 h of hypoxia(hypoxia1h,dissolved oxygen(DO)=1.5±0.1 mg L^(−1)),4h of hypoxia(hypoxia4h,DO=1.5±0.1 mg L^(−1)),4h of reoxygen(reoxygen4h,DO=8.0±0.2 mg L^(−1))after 4h of hypoxia(DO=1.5 mg L^(−1))and normoxia or control(DO=8.0±0.2 mg L^(−1))groups.The results showed that a total of 3068 genes were identified as differentially expressed genes(DEGs)based on the criteria∣log2(Fold change)∣>1.0 and adjusted P-value<0.05.A total of 7761141 and 1151 DEGs were obtained from hypoxia1h,hypoxia4h and reoxygen4h groups,respectively.The enrichment pathway analysis showed that the DEGs were significantly enriched in ribosome biogenesis in eukaryotes,retinol metabolism,DNA replication and the oxidative phosphorylation(OXPHOS)pathways.Thirteen DEGs from the RNA-seq results were validated by quantitative real-time polymerase chain reaction(qRT-PCR).These candidate genes are considered as important regulatory factors involved in the hypoxia stress response in S.sihama.

低氧胁迫对绿鳍马面鲀鳃组织抗氧化酶、呼吸相关酶及磷酸酶活性的影响

为研究低氧胁迫下绿鳍马面鲀(Thamnaconus septentrionalis)鳃组织的应激响应机制,进行了溶解氧水平分别为8、5、2 mg/L的低氧胁迫试验(其中8 mg/L组为对照组,其余2组为低氧组),分析不同溶解氧条件下其鳃组织中酶活性的变化。试验结果显示,随着溶解氧水平的降低,绿鳍马面鲀鳃组织中过氧化氢酶(CAT)和超氧化物歧化酶(SOD)活性和总抗氧化能力(T-AOC)均呈现上升趋势;丙二醛(MDA)含量与对照组相比降低;乳酸脱氢酶(LDH)活性随着溶解氧的降低而升高;酸性磷酸酶(ACP)活性随着溶解氧的下降呈现先上升后下降的趋势,碱性磷酸酶(AKP)活性则呈现上升的趋势。试验结果表明,低氧胁迫能够影响绿鳍马面鲀鳃组织内抗氧化酶、呼吸相关酶及磷酸酶的活性,在低氧胁迫过程中,绿鳍马面鲀会通过调节抗氧化酶的活性来激活体内抗氧化防御系统,从而保护机体免受缺氧导致的氧化损伤。

High-altitude hypoxia induces disorders of the brain- endocrine-immune network through activation of corticotropin-releasing factor and its type- 1 receptors

High-altitude hypoxia can induce physiological dysfunction and mountain sickness,but the underlying mechanism is not fully understood.Corticotrophin-releasing factor(CRF) and CRF type-1 receptors(CRFR1) are members of the CRF family and the essential controllers of the physiological activity of the hypothalamo-pituitary-adrenal(HPA) axis and modulators of endocrine and behavioral activity in response to various stressors.We have previously found that high-altitude hypoxia induces disorders of the brain-endocrine-immune network through activation of CRF and CRFR1 in the brain and periphery that include activation of the HPA axis in a time-and dose-dependent manner,impaired or improved learning and memory,and anxiety-like behavioral change.Meanwhile,hypoxia induces dysfunctions of the hypothalamo-pituitary-endocrine and immune systems,including suppression of growth and development,as well as inhibition of reproductive,metabolic and immune functions.In contrast,the small mammals that live on the Qinghai-Tibet Plateau alpine meadow display low responsiveness to extreme high-altitudehypoxia challenge,suggesting well-acclimatized genes and a physiological strategy that developed during evolution through interactions between the genes and environment.All the findings provide evidence for understanding the neuroendocrine mechanisms of hypoxia-induced physiological dysfunction.This review extends these findings.

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.

低氧联合酸胁迫红小豆/绿豆萌发富集GABA及富含GABA芽豆复配米饭的工艺优化

为了研究低氧联合酸胁迫对红小豆和绿豆GABA富集的作用,采用单因素对萌发时间、萌发温度、低氧时间和L-谷氨酸浓度进行考察,在确定高GABA芽豆的胁迫条件基础上,将富含GABA红小豆、绿豆与大米进行复配,利用D-混料设计优化芽豆米饭配方工艺。结果显示,低氧联合酸胁迫对红小豆、绿豆富集GABA有积极促进作用,在萌发时间48 h、萌发温度40℃、低氧时间15 h和L-谷氨酸浓度2.5 mg/mL条件下,萌发红小豆中GABA高达158.32±3.24 mg/100 g。绿豆胁迫条件为萌发时间24 h、萌发温度35℃、低氧时间15 h和L-谷氨酸浓度2.5 mg/mL时,其中GABA含量最高为141.57±4.35 mg/100 g。在此基础上,通过D-混料设计优化确定了复配芽豆米饭的最佳配方为:大米76%、萌发绿豆11%、萌发红小豆13%,此条件下,芽豆米饭GABA含量为23.73±1.03 mg/100 g,感官评分均值为88.76±2.47,制得的芽豆米饭口感、色泽、香味均在可接受范围内,且积累了GABA活性成分,提升了芽豆米饭的营养及功能特性,为进一步开发杂粮复配米饭提供理论参考。

Effects of transfected adenovirus-mediated transcription factor X-box binding protein 1 on hippocampal-derived neural stem cell proliferation and apoptosis under hypoxia

BACKGROUND： Neural stem cell （NSC） survival is closely associated with cell apoptosis in ischemic-hypoxic regions following transplantation. Numerous studies have revealed that X-box binding protein 1 （XBP1） is a transcription factor during endoplasmic reticulum unfolded protein response and is essential for cell survival, differentiation, and anti-apoptotic effects. OBJECTIVE： To determine the effects of the XBP1 gene on NSC proliferation and apoptosis under hypoxic conditions following XBP1 gene transfection into rat embryonic hippocampal NSCs using recombinant adenovirus vector. DESIGN, TIME AND SETTING： In vitro experiments were performed at the Laboratory of Cell Biology of Jilin University and Laboratory of Proteomics, Department of Neurology, Jilin University China from September 2008 to November 2009. MATERIALS： Recombinant adenovirus package XBP1 gene and Ad-XBPl-enhanced green fluorescent protein plasmid （Guangzhou Easywin BioMed Technology, China）, rabbit anti-XBP1 and its target gene estrogen receptor degradation-enhancing a-mannosidase-like protein （EDEM） glucose-regulated protein 78 （GRP78）, anti-apoptotic molecule Bcl-2 and proapoptotic molecule Bax polyclonal antibody （Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA）, and COCI2 （Sigma, St. Louis, MO, USA） were used in the present study. METHODS： Hippocampi from embryonic, Sprague Dawley rats on gestational day 16 were harvested for NSC isolation and cloning, followed by immunofluorescence for Nestin and sub-culturing. The recombinant adenovirus Ad-XBPl-enhanced green fluorescent protein plasmid was transfected into rat embryonic hippocampal NSCs, and then CoCl2 was applied to induce hypoxia. MAIN OUTCOME MEASURES： Cell quantification and 3-（4, 5-dimethylthiazol-2-yl）-2, 5-diphenyltetrazolium bromide colorimetric assay were utilized to detect proliferation in XBPl-transfected NSCs for 7 consecutive days. Western blot assay was utilized to quantify XBP1 GRP78, EDEM, Bcl-2, and Bax expression. Flow cytometry was used to measure apoptosis. RESULTS： NSC proliferation was significantly enhanced following XBP1 gene transfection （P 〈 0.05）. Under hypoxic conditions, GRP78, EDEM, and Bcl-2 levels increased, but Bax levels decreased. In addition, NSC apoptosis decreased following transfection （P 〈 0.05）. CONCLUSION： The XBP1 gene was successfully transfected into rat embryonic hippocampal NSCs using a recombinant adenovirus vector. NSC proliferation following transfection, as well as anti-apoptotic effects under hypoxia, was significantly increased.

Effect of safflower injection on endoplasmic reticulum stress-induced apoptosis in rats with hypoxic pulmonary hypertension

Objective To explore the effects of safflower injection on prevention and treatment of hypoxic pulmonary hypertension and clarify the function of the endoplasmic reticulum stress apoptosis pathway during the process.Methods Thirty male SD rats were randomly grouped as normal control group,hypoxiahypercapnia group and hypoxia+safflower group.The latter two groups were put in the cabin with oxygen concentration ranged from 9% to 11% and carbon dioxide concentration from 5% to 6%.The pulmonary artery pressure and the index of right ventricular hypertrophy were determined after hypoxia exposure(8 h/d×28 d).Changes in morphology of lung tissue were observed by electron microscopy.To explore the possible mechanisms,we also detected apoptosis and apoptosis-related genes/proteins in lung tissue by TUNEL reactivity and PCR and Western blot.Results Compared with the normal control group,pulmonary artery pressure and the index of right ventricular hypertrophy in hypoxia group were 45% and 33.4% higher,respectively.Tiny blood vessel wall of lungs was thickened and edema,and proliferation of collagen fibers was obvious under the electron microscope.TUNEL staining of apoptotic cells in lung tissues showed more high brightness green fluorescence(+～++),but less green fluorescence showed in the pulmonary vascular smooth muscle cell layer,and apoptosis index(AI) value was 150% higher;gene and protein expression levels of endoplasmic reticulum stress pathway were increased.Compared with hypoxia-hypercapnia group,pulmonary artery pressure and the index of right ventricular hypertrophy in the hypoxia+safflower group were 18% and 15.6% lower,respectively;collagen fibers were decreased,and smooth muscle cells and epithelial cells were got apoptotic-like changes under the electron microscope.TUNEL staining of apoptotic cells in lung tissues showed brighter green fluorescence(++～+++);the high brightness green fluorescence showed in pulmonary vascular smooth muscle cell layer,and apoptotic index(AI) value was 40% higher;gene and protein expressions of endoplasmic reticulum stress pathway were significantly upregulated.Conclusion Our findings demonstrate that safflower injection could activate endoplasmic reticulum stressinduced apoptosis and especially promote apoptosis in pulmonary vascular smooth muscle cells.