DI-3-n-butylphthalide exerts neuroprotective effects by modulating hypoxia-inducible factor 1-alpha ubiquitination to attenuate oxidative stress-induced apoptosis

DI-3-n-butylphthalide is used to treat mild and moderate acute ischemic stroke.However,the precise underlying mechanism requires further investigation.In this study,we investigated the molecular mechanism of DI-3-n-butylphthalide action by various means.We used hydrogen peroxide to induce injury to PC12cells and RAW264.7 cells to mimic neuronal oxidative stress injury in stroke in vitro and examined the effects of DI-3-n-butylphthalide.We found that DI-3-nbutylphthalide pretreatment markedly inhibited the reduction in viability and reactive oxygen species production in PC12 cells caused by hydrogen peroxide and inhibited cell apoptosis.Furthermore,DI-3-n-butylphthalide pretreatment inhibited the expression of the pro-apoptotic genes Bax and Bnip3.DI-3-nbutylphthalide also promoted ubiquitination and degradation of hypoxia inducible factor 1α,the key transcription factor that regulates Bax and Bnip3 genes.These findings suggest that DI-3-n-butylphthalide exhibits a neuroprotective effect on stroke by promoting hypoxia inducible factor-1α ubiquitination and degradation and inhibiting cell apoptosis.

Human umbilical cord mesenchymal stem cells derivedexosomes on VEGF-A in hypoxic-induced mice retinal astrocytes and mice model of retinopathy of prematurity

AIM:To observe the effect of human umbilical cord mesenchymal stem cells(hUCMSCs)secretions on the relevant factors in mouse retinal astrocytes,and to investigate the effect of hUCMSCs on the expression of vascular endothelial growth factor-A(VEGF-A)and to observe the therapeutic effect on the mouse model of retinopathy of prematurity(ROP).METHODS:Cultured hUCMSCs and extracted exosomes from them and then retinal astrocytes were divided into control group and hypoxia group.MTT assay,flow cytometry,reverse transcription-polymerase chain reaction(RT-PCR)and Western blot were used to detect related indicators.Possible mechanisms by which hUCMSCs exosomes affect VEGF-A expression in hypoxia-induced mouse retinal astrocytes were explored.At last,the efficacy of exosomes of UCMSCs in a mouse ROP model was explored.Graphpad6 was used to comprehensively process data information.RESULTS:The secretion was successfully extracted from the culture supernatant of hUCMSCs by gradient ultracentrifugation.Reactive oxygen species(ROS)and hypoxia inducible factor-1α(HIF-1α)of mice retinal astrocytes under different hypoxia time and the expression level of VEGF-A protein and VEGF-A mRNA increased,and the ROP cell model was established after 6h of hypoxia.The secretions of medium and high concentrations of hUCMSCs can reduce ROS and HIF-1α,the expression levels of VEGF-A protein and VEGF-A mRNA are statistically significant and concentration dependent.Compared with the ROP cell model group,the expression of phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)/mammalian target of rapamycin(mTOR)signal pathway related factors in the hUCMSCs exocrine group is significantly decreased.The intravitreal injection of the secretions of medium and high concentrations of hUCMSCs can reduce VEGF-A and HIF-1αin ROP model tissues.HE staining shows that the number of retinal neovascularization in ROP mice decreases with the increase of the dose of hUCMSCs secretion.CONCLUSION:In a hypoxia induced mouse retinal astrocyte model,hUCMSCs exosomes are found to effectively reduce the expression of HIF-1αand VEGF-A,which are positively correlated with the concentration of hUCMSCs exosomes.HUCMSCs exosomes can effectively reduce the number of retinal neovascularization and the expression of HIF-1αand VEGF-A proteins in ROP mice,and are positively correlated with drug dosage.Besides,they can reduce the related factors on the PI3K/AKT/mTOR signaling pathway.

Vascular endothelial growth factor induced angiogenesis following focal cerebral ischemia/reperfusion injury in rabbits

BACKGROUND： Therapeutic angiogenesis has opened up new pathway for the treatment of ischemic cerebrovascular disease in recent years. The exploration of the effect of vascular endothelial growth factor （VEGF） on inducing angiogenesis following ischemia/reperfusion injury can provide better help for the long-term treatment of cerebrovascular disease in clinic. OBJECTIVE： To observe the effect of VEGF on inducing angiogenesis following focal cerebral ischemia /reperfusion injury in rabbits through the angiogenesis of microvessels reflected by the expression of the factors of vascular pseudohemophilia. DESIGN： A randomized controlled animal tria SETTNG： Department of Medical Imaging, Second Hospital of Hebei Medical University MATERIALS： Sixty-five healthy male New Zealand rabbits of clean degree, weighing （2.6±0.2） kg, aged 4.5-5 months, were used. The polyclonal antibody against vascular pseudohemophilia （Beijing Zhongshan Company）, recombinant VEGF165 （Peprotech Company, USA）, biotinylated second antibody and ABC compound （Wuhan Boster Company） were applied. METHODS： The experiments were carried out in the Laboratory of Neuromolecular Imaging and Neuropathy, Second Hospital of Hebei Medical University from May to August in 2005. （1） The rabbits were randomly divided into three groups： sham-operated group （n=15）, control group （n=25） and VEGF-treated group （n=-25）. In the control group and VEGF-treated group, models were established by middle cerebral artery occlusion （MCAO） induced focal cerebral ischemia/reperfusion. In the VEGF-treated group, VEGF165 （2.5 mg/L） was stereotactically injected into the surrounding regions of the infarcted sites immediately after the 2-hour ischemia/reperfusion; Saline of the same dosage was injected in the control group. But the rabbits in the sham-operated group were only drilled but not administrated. （2） The experimental indexes were observed on the 3^rd 7^th, 14^th, 28^th and 70^th days of the experiment respectively, 3 rabbits in the sham-operated group and 5 in the control group and VEGF-treated group were observed at each time point. The brain tissues in the surrounding regions of the infarcted sites were collected. The positive expressions of the factors of vascular pseudohemophilia in vascular endothelial cells were analyzed with immunohistochemical method. The microvessels in unit statistical field were counted with the imaging analytical software. MAIN OUTCOME MEASURES： The changes of microvascular density in the brain tissue and the positive expressions of the factors of vascular pseudohemophilia in the surrounding regions of the infarcted sites were observed on the 3^rd 7^th, 14^th, 28^th and 70^th days of the experiment. RESULTS： All the 65 New Zealand rabbits were involved in the analysis of results without deletion. Changes of the number of microvessels at different time points in each group： There were no obvious changes at different time points in the sham-operated group. The numbers of microvessels at 7 and 14 days were obviously more in the control group than in the sham-operated group [（6.0±1.1）, （9.0±0.9） microvessels; （3.0±1.1）, （3.0±1.1） microvessels; P〈 0.05-0.01], and those at 3, 7, 14 and 28 days were obviously more in the VEGF-treated group than in the control group [（8.3±2.0）, （13.4±1.4）, （15.5±2.3）, （6.8± 1.0） microvessels; （3.4±0.6）, （6.0±1.1）, （9.0±0.9）, （3.2±0.8） microvessels; P 〈 0.01]. （2） Positive expressions of the factors of vascular pseudohemophilia in the surrounding regions of infarcted sites： There were no obvious changes at different time points in the sham-operated group. In the control group, the changing law of the expressions was the same as that for the number of microvessels that the expression began to mildly increase at 7 days, reached the peak value at 14 days, and began to reduce at 28 days. In the VEGF-treated group, the expression was obviously increased at 3 days, also reached the peak value at 14 days, and reduced to the normal level at 70 days, but the expressions were obviously stronger than those in the control group at the same time points. CONCLUSION： Angiogenesis can be obviously induced in rabbits after the focal cerebral ischemia/reperfusion injury is treated with VEGF for 18 days.

Microthrombosis induced by mechanical factors and light-dye treatment

Introduction Thrombosis is the formation of a blood clot in a blood vessel. When thrombosis happens in the brain,it would cause stroke; when happens in the heart,it would cause heart attack. If a thrombus breaks and travels to the lung,it would

The role of nerve growth factor inducible protein B in the pathogenesis of levodopa-induced dyskinesias

Objective： To study the role of the expression of nerve growth factor inducible protein B gene （NGFI-B） in striatum in the pathogenesis of levodopa-induced dyskinesias （LID）. Methods： The rat model of LID was treated with SCH 23390（1 mg/kg ip,a dopamine D1 antagonist） and haloperidol （1 mg/kg ip,a dopanfme D2 antagonist） respectively. Reverse transcriptase-polymerase chain reaction （RT-PCR） was used to measure the expression of NGFI-B mRNA in stiiatam and the behavior changes were observed. Resuits： After treatment with SCH23390, abnormal involuntary movement （AIM） in LID rats was decreased （ P 〈 0.05 ） and the expression of NGFI-B mRNA in striatum did not change significantly. After treatment with haloperidol, the changes of AIM in LID rats were not significant and the expression of NGFI-B mRNA was increased significantly（ P 〈 0.01）. Conclusion： LID is associated with over-expression of NGFI-B in striatum. Abnormal activity in the direct pathway and the basal ganglia circuit could be involved in the occurrence of LID.

Targeted anti-cancer therapy: Co-delivery of VEGF siRNA and Phenethyl isothiocyanate (PEITC) via cRGD-modified lipid nanoparticles for enhanced anti-angiogenic efficacy

Anti-tumor angiogenesis therapy, targeting the suppression of blood vessel growth in tumors, presents a potent approach in the battle against cancer. Traditional therapies have primarily concentrated on single-target techniques, with a specific emphasis on targeting the vascular endothelial growth factor, but have not reached ideal therapeutic efficacy. In response to this issue, our study introduced a novel nanoparticle system known as CS-siRNA/PEITC&L-cRGD NPs. These chitosan-based nanoparticles have been recognized for their excellent biocompatibility and ability to deliver genes. To enhance their targeted delivery capability, they were combined with a cyclic RGD peptide (cRGD). Targeted co-delivery of gene and chemotherapeutic agents was achieved through the use of a negatively charged lipid shell and cRGD, which possesses high affinity for integrin αvβ3 overexpressed in tumor cells and neovasculature. In this multifaceted approach, co-delivery of VEGF siRNA and phenethyl isothiocyanate (PEITC) was employed to target both tumor vascular endothelial cells and tumor cells simultaneously. The co-delivery of VEGF siRNA and PEITC could achieve precise silencing of VEGF, inhibit the accumulation of HIF-1α under hypoxic conditions, and induce apoptosis in tumor cells. In summary, we have successfully developed a nanoparticle delivery platform that utilizes a dual mechanism of action of anti-tumor angiogenesis and pro-tumor apoptosis, which provides a robust and potent strategy for the delivery of anti-cancer therapeutics.

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.

Noise exposure induced cochlear hair cell death pathways in guinea pig

Objective To understand the mechanism of noise exposure induced outer hair cells(OHCs) death pathways. Methods Thirty two guinea pigs were used in this study. The animals were either exposed for 4 h/day to broadband noise at 122 dB SPL (A-weighted) for 2 consecutive days or perfused with MNNG. After auditory test, the cochleae of animals were dissected. Propidium iodide (PI), a DNA intercalating fluorescent probe, was used to trace morphological changes in OHC nuclei. F-actin staining was used to determine missing OHCs. Caspase-3 was detected in living organ of Corti whole mounts using the fluorescent probe. The single strand DNA (ssDNA) in apoptotic OHCs in guinea pigs and apoptosis inducing factor (AIF) in hair cells in guinea pigs were examined by immunohistology method. Whole mounts of organ of Corti were prepared. Morphological and fluorescent changes were examined under a confocal microscope. Results (1) Both apoptotic and necrotic hair cells appeared following noise exposure. (2) Noise exposure induced single strand DNA in apoptotic OHCs but not in the normal OHCs. (3) Either after noise exposure or after MNNG perfusion, apoptotic OHCs were featured by nuclear condensation or fragmentation with caspase-3 activation, whereas necrotic OHCs were characterized by nuclear swelling without caspase-3 activation. (4) In normal organ of Corti, AIF was located in the mitochondria areas. After noise exposure, AIF was translocated from mitochondria in apoptotic and necrotic OHCs. Conclusion These findings indicate that noise exposure damages DNA in the OHC, which triggers action of Caspase-3. Subsequently, AIF is translocated to the nucleus, leading to DNA damage and OHCs death.

Role of apoptosis-inducing factor in perinatal hypoxic-ischemic brain injury

Perinatal complications,such as asphyxia,can cause brain injuries that are often associated with subsequent neurological deficits,such as cerebral palsy or mental retardation.The mechanisms of perinatal brain injury are not fully understood,but mitochondria play a prominent role not only due to their central function in metabolism but also because many proteins with apoptosis-related functions are located in the mitochondrion.Among these proteins,apoptosis-inducing factor has already been shown to be an important factor involved in neuronal cell death upon hypoxia-ischemia,but a better understanding of the mechanisms behind these processes is required for the development of more effective treatments during the early stages of perinatal brain injury.In this review,we focus on the molecular mechanisms of hypoxic-ischemic encephalopathy,specifically on the importance of apoptosis-inducing factor.The relevance of apoptosis-inducing factor is based not only because it participates in the caspase-independent apoptotic pathway but also because it plays a crucial role in mitochondrial energetic functionality,especially with regard to the maintenance of electron transport during oxidative phosphorylation and in oxidative stress,acting as a free radical scavenger.We also discuss all the different apoptosis-inducing factor isoforms discovered,focusing especially on apoptosis-inducing factor 2,which is only expressed in the brain and the functions of which are starting now to be clarified.Finally,we summarized the interaction of apoptosis-inducing factor with several proteins that are crucial for both apoptosis-inducing factor functions(prosurvival and pro-apoptotic)and that are highly important in order to develop promising therapeutic targets for improving outcomes after perinatal brain injury.

Hypoxia upregulates hypoxia inducible factor(HIF)-3α expression in lung epithelial cells: characterization and comparison with HIF-1α

The role of the hypoxia-inducible factor(HIF)subunits 1α and 2α in response to hypoxia is well established in lungepithelial cells,whereas little is known about HIF-3α with respect to transcriptional and translational regulation by hy-poxia.HIF-3α and HIF-1α are two similar but distinct basic helix-loop-helix-PAS proteins,which have been postulatedto activate hypoxia responsive genes in response to hypoxia.Here,we used quantitative real time RT-PCR and immu-noblotting to determine the activation of HIF-3α vs.HIF-1α by hypoxia.HIF-3α was strongly induced by hypoxia(1%O_2)both at the level of protein and mRNA due to an increase in protein stability and transcriptional activation,whereasHIF-1α protein and mRNA levels enhanced transiently and then decreased because of a reduction in its mRNA stabilityin A549 cells,as measured on mRNA and protein levels.Interestingly,HIF-3α and HIF-1α exhibited strikingly similarresponses to a variety of activating or inhibitory pharmacological agents.These results demonstrate that HIF-3α is ex-pressed abundantly in lung epithelial cells,and that the transcriptional induction of HIF-3α plays an important role in theresponse to hypoxia in vitro.Our findings suggest that HIF-3α,as a member of the HIF system,is complementary ratherthan redundant to HIF-1α induction in protection against hypoxic damage in alveolar epithelial cells.

Expression of caspase-3 and hypoxia inducible factor 1αin hepatocellular carcinoma complicated by hemorrhage and necrosis

BACKGROUND Hepatocellular carcinoma(HCC)is a malignant tumor that occurs in the liver.Its onset is latent,and it shows high heterogeneity and can readily experience intrahepatic metastasis or systemic metastasis,which seriously affects patients’quality of life.Numerous studies have shown that hypoxia inducible factor1α(HIF-1α)plays a significant role in the occurrence and development of tumors,as it promotes the formation of intratumoral vessels and plays a key role in their metastasis and invasion.Some studies have reported that caspase-3,which is induced by various factors,is involved in the apoptosis of tumor cells.AIM To investigate the expression of caspase-3 and HIF-1αand their relationship to the prognosis of patients with primary HCC complicated by pathological changes of hemorrhage and necrosis.METHODS A total of 88 patients with HCC complicated by pathological changes of hemorrhage and necrosis who were treated at our hospital from January 2017 to December 2019 were selected.The expression of caspase-3 and HIF-1αin HCC and paracancerous tissues from these patients was assessed.RESULTS The positive expression rate of caspase-3 in HCC tissues was 27.27%,which was significantly lower than that in the paracancerous tissues(P<0.05),while the positive expression rate of HIF-1αwas 72.73%,which was significantly higher than that in the paracancerous tissues(P<0.05).The positive expression rates for caspase-3 in tumor node metastasis(TNM)stage III and lymph node metastasis tissues were 2.78%and 2.50%,respectively,which were significantly lower than those in TNM stage I-II and non-lymph node metastasis tissues(P<0.05).The positive expression rates of HIF-1αin TNM stage III,lymph node metastasis,and portal vein tumor thrombus tissues were 86.11%,87.50%,and 88.00%,respectively,and these values were significantly higher than those in TNM stage I-II,non-lymph node metastasis,and portal vein tumor thrombus tissues(P<0.05).The expression of caspase-3 and HIF-1αin HCC tissues were negatively correlated(rs=−0.426,P<0.05).The median overall survival time of HCC patients was 18.90 mo(95%CI:17.20–19.91).The results of the Cox proportional risk regression model analysis showed that TNM stage,portal vein tumor thrombus,lymph node metastasis,caspase-3 expression,and HIF-1αexpression were the factors influencing patient prognosis(P<0.05).CONCLUSION The expression of caspase-3 decreases and HIF-1αincreases in HCC tissues complicated by pathological changes of hemorrhage and necrosis,and these are related to clinicopathological features and prognosis.

Chloride channel blocker 4,4-diisothiocyanatostilbene-2,2'-disulfonic acid inhibits nitric oxide-induced apoptosis in cultured rat hippocampal neurons

Apoptosis in cultured rat hippocampal neurons was induced using the nitric oxide donor 3-morpholinosydnonimine, and cells were treated with the chloride channel blocker, 4,4- diisothiocyanatostilbene-2,2＇-disulfonic acid. Results showed that the survival rate of neurons was significantly increased after treatment with 4,4-diisothiocyanatostilbene-2,2＇-disulfonic acid, and the rate of apoptosis decreased. In addition, the expression of the apoptosis-related proteins poly（adenosine diphosphate-ribose）polymerase-1 and apoptosis-inducing factor were significantly reduced. Our experimental findings indicate that the chloride channel blocker 4,4- diisothiocyanatostilbene-2,2＇-disulfonic acid can antagonize apoptotic cell death of hippocampal neurons by inhibiting the expression of the apoptosis-related proteins poly（adenosine diphosphate-ribose）polymerase-1 and apoptosis-inducing factor.

Evaluation of hypoxia inducible factor targeting pharmacological drugs as antileishmanial agents

Objective:To evaluate whether hypoxia inducible factor(HIF-1α) targeting pharmacological drugs,echinomycin,resveratrol and CdCl_2 which inhibit HIF-1α stimulation,and mimosine,which enhances the stability of HIF-1α present antileishmanial properties.Methods:The leishmanicidal effect of drugs was evaluated in mouse macrophages and Balb/c mouse model for cutaneous leishmaniosis.Results:Resveratrol and CdCl_2 reduced the parasite load [IC50,(27.3±2.25) μM and(24.8±0.95) μM,respectively].The IC50 value of echinomycin was(22.7±7.36) nM and mimosine did not alter the parasite load in primary macrophages.The macrophage viability IC50 values for resveratrol,echinomycin and CdCl_2 and mimosine were >40 μM,>100 nM,> 200 μM and>2 000 μM,respectively.In vivo no differences between cutaneous lesions from control,resveratrol-and echinomycin-treated Balb/c mice were detected.Conclusions:Resveratrol,echinomycin and CdCl_2 reduce parasite survival in vitro.The HIF-1α targeting pharmacological drugs require further study to more fully determine their anti-Leishmania potential and their role in therapeutic strategies.

Sirtuin1 attenuates acute liver failure by reducing reactive oxygen species via hypoxia inducible factor 1α

BACKGROUND The occurrence and development of acute liver failure(ALF)is closely related to a series of inflammatory reactions,such as the production of reactive oxygen species(ROS).Hypoxia inducible factor 1α(HIF-1α)is a key factor that regulates oxygen homeostasis and redox,and the stability of HIF-1αis related to the ROS level regulated by Sirtuin(Sirt)family.The activation of Sirt1 will lead to a powerful antioxidant defense system and therapeutic effects in liver disease.However,little is known about the relationship between HIF-1αand Sirt1 in the process of ALF and the molecular mechanism.AIM To investigate whether HIF-1αmay be a target of Sirt1 deacetylation and what the effects on ALF are.METHODS Mice were administrated lipopolysaccharide(LPS)/D-gal and exposed to hypoxic conditions as animal model,and resveratrol was used as an activator of Sirt1.The cellular model was established with L02 cells stimulated by LPS.N-acetyl-Lcysteine was used to remove ROS,and the expression of Sirt1 was inhibited by nicotinamide.Western blotting was used to detect Sirt1 and HIF-1αactivity and related protein expression.The possible signaling pathways involved were analyzed by immunofluorescent staining,co-immunoprecipitation,dihydroethidium staining,and Western blotting.RESULTS Compared with mice stimulated with LPS alone,the expression of Sirt1 decreased,the level of HIF-1αacetylation increased in hypoxic mice,and the levels of carbonic anhydrase 9 and Bcl-2-adenovirus E1B interacting protein 3 increased significantly,which was regulated by HIF-1α,indicating an increase of HIF-1αactivity.Under hypoxia,the down-regulation of Sirt1 activated and acetylated HIF-1αin L02 cells.The inhibition of Sirt1 significantly aggravated this effect and the massive production of ROS.The regulation of ROS was partly through peroxisome proliferatoractivated receptor alpha or AMP-activated protein kinase.Resveratrol,a Sirt1 activator,effectively relieved ALF aggravated by hypoxia,the production of ROS,and cell apoptosis.It also induced the deacetylation of HIF-1αand inhibited the activity of HIF-1α.CONCLUSION Sirt1 may have a protective effect on ALF by inducing HIF-1α deacetylation to reduce ROS.

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.

Mammalian target of rapamycin complex 1 as an inducer of neurotrophic factors in dopaminergic neurons

The defining neuropathological feature of Parkinson＇s disease （PD） is the loss of nigrostriatal dopaminergic （DA） projections. This results in striatal dopamine levels and a biochemical reduction of movement disorders, such as a tremor at rest, rigidity of the limbs, bradykinesia, and postural instability （Kim et al., 2011; Kim et al., 2012; Burke and O＇Malley, 2013; Leem et al., 2014; Namet al., 2014）.

Expression of tumor necrosis factor related apoptosis inducing ligand receptor in glioblastoma

BACKGROUND： Receptors for tumor necrosis factor related apoptosis inducing ligand （TRAIL） include death receptor 4, death receptor 5, decoy receptor 1, and decoy receptor 2. Activation of death receptor 4 and 5 selectively kills tumor cells. OBJECTIVE： To detect TRAIL receptor expression in glioblastoma by immunohistochemistry and RT-PCR, and to compare this expression to that in normal brain tissue. DESIGN： Observational analysis. SETTING： Department of Pathology, the First Affiliated Hospital of Zhengzhou University; Henan Tumor Pathology Key Laboratory. PARTICIPANTS： Twenty-five patients （17 males and 8 females） who received glioblastoma resection were selected from the Fifth Affiliated Hospital of Zhengzhou University, between September 2003 to June 2004. All glioblastoma samples were diagnosed pathologically. Twenty patients （12 males and 8 females） with craniocerebral injury who received normal brain tissue resection were selected in the same time period. There were no significant differences in sex and age between glioblastoma patients or between craniocerebral injury patients （P 〉 0.05）. All patients and appropriate relatives provided informed consent, and this study was approved by the local research ethics committee. METHODS： Polyclonal antibody against TRAIL receptors and an immunohistochemical kit （batch number： 200502） were purchased from Boster Company, Wuhan. Immunohistochemistry： Expression of death receptor 4, death receptor 5, decoy receptor l, and decoy receptor 2 were observed in both glioblastoma and normal brain tissue. The experiment was performed according to the kit instructions, and positive staining was brown-yellow. Assessment： There were no positive signals （-）; weakly positive signals, positive cells 〈 25% （＋）; weakly positive signals, positive cells 25%-50% （＋＋）; strongly positive signals, positive cells 50%-75% （＋＋＋）; strongly positive signals, positive cells 〉 75% （＋＋＋＋）. Evaluation： Expression levels of TRAIL receptors were estimated in both normal brain tissue and glioblastoma. Expression of decoy receptor 1 and decoy receptor 2 mRNA in glioblastoma were detected by reverse transcription polymerase chain reaction, and expression of decoy receptor in glioblastoma was estimated. MAIN OUTCOME MEASURES： Comparison of death receptor and decoy receptor protein expression between glioblastoma and normal brain tissue; decoy receptor mRNA expression in glioblastoma. RESULTS： Death receptor protein expression was strongly positive （＋＋＋） in glioblastoma, while it was weakly positive （＋, ＋＋） in normal brain tissue. Therefore, expression rate of death receptor protein in the glioblastoma was significantly higher than that in the normal brain tissue （.~ 2 = 18.48, 23.03, P 〈 0.01）. Decoy receptor protein expression in the glioblastoma was significantly lower than that in the normal brain tissue （ x2 = 6.65, 18.76, P 〈 0.01）. The level of decoy receptor mRNA expression in glioblastoma was significantly higher than those of protein expression （ x 2 = 9.82, 10.09, P〈 0.01）. CONCLUSION： High expression of death receptor and low expression of decoy receptor are frequently observed in glioblastoma, suggesting that TRAIL receptor genes show an anti-tumor and expressive response during the initiation and development of the tumor. There are significant differences in decoy receptor expression between normal brain tissue and glioblastoma, suggesting that the restricted expression of decoy receptor in glioblastoma is regulated at the post-transcriptional level.

Maternal Lead Exposure Induces Down-regulation of Hippocampal Insulin-degrading Enzyme and Nerve Growth Factor Expression in Mouse Pups

Lead exposure is a known potential risk factor for neurodegenerative diseases such as Alzheimer’s disease （AD）. Exposure to lead during the critical phase of brain development has been linked with mental retardation and hypophrenia in later life.

Synergistic effects of brain-derived neurotrophic factor and retinoic acid on inducing the differentiation of bone marrow stromal cells into neuron-like cells in adult rats in vitro

BACKGROUND： Under induction of retinoic acid （RA）, bone marrow stromal cells （BMSCs） can differentiate into nerve cells or neuron-like cells, which do not survive for a long time, so those are restricted to an application. Other neurotrophic factors can also differentiate into neuronal cells through inducing BMSCs; especially, brain-derived neurotrophic factor （BDNF） can delay natural death of neurons and play a key role in survival and growth of neurons. The combination of them is beneficial for differentiation of BMSCs. OBJECTIVE： To investigate the effects of BDNF combining with RA on inducing differentiation of BMSCs to nerve cells of adult rats and compare the results between common medium group and single BDNF group. DESIGN： Randomized controlled animal study SETTING： Department of Neurology, Affiliated Hospital of Xuzhou Medical College MATERIALS： The experiment was carried out in the Clinical Neurological Laboratory of Xuzhou Medical College from September 2003 to April 2005. A total of 24 SD rats, of either gender, 2 months old, weighing 130-150 g, were provided by Experimental Animal Center of Xuzhou Medical College [certification： SYXK （su） 2002-0038]. Materials and reagents： low-glucose DMEM medium, bovine serum, BDNF, RA, trypsin, separating medium of lymphocyte, monoclonal antibody of mouse-anti-nestin, neuro-specific enolase, glial fibrillary acidic protein （GFAP） antibody, SABC kit, and diaminobenzidine （DAB） color agent. All these mentioned above were mainly provided by SIGMA Company, GIBCO Company and Boshide Company. METHODS： Bone marrow of SD rats was selected for density gradient centrifugation. BMSCs were undertaken primary culture and subculture; and then, those cells were induced respectively in various mediums in total of 3 groups, including control group （primary culture）, BDNF group （20 μg/L BDNF） and BDNF＋RA group （20 μg/L BDNF plus 20 μg/L RA）. On the 3^rd and the 7^th days after induction, BMSCs were stained immunocytochemically with nestin （sign of nerve stem cells）, neuron-specific enolase （NSE, sign of diagnosing neurons） and GFAP （diagnosing astrocyte）, and evaluated cellular property. MAIN OUTCOME MEASURES ： Induction and differentiation in vitro of BMSCs in 3 groups RESULTS： （1） Induction and differentiation of BMSCs： Seven days after induction, cells having 2 or more apophyses were observed. Soma shaped like angle or erose form, which were similar to neurons and glial cells having strong refraction. （2） Results of immunocytochemical detection： Three days after induction, rate of positive cells in BDNF＋RA group was higher than that in BDNF group and control group [（86.15±4.58）%, （65.43±4.23）%, （4.18±1.09）%, P 〈 0.01]. Seven days after induction, rate of positive cells was lower in BDNF group and BDNF＋RA group than that in both groups at 3 days after induction [（31.12±3.18）%, （29.35±2.69）%, P 〈 0.01]; however, amounts of positive cells of NSE and GFAP were higher than those at 3 days after induction （P 〈 0.01）; meanwhile, the amount in BDNF＋RA group was remarkably higher than that in BDNF group （P 〈 0.01）. CONCLUSION： Combination of BDNF and RA can cooperate differentiation of BMSCs into neurons and astrocyte, and the effect is superior to single usage of BDNF.

Mechanisms of regulation of PFKFB expression in pancreatic and gastric cancer cells

Enzymes 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 and -4 (PFKFB-3 and PFKFB-4) play a significant role in the regulation of glycolysis in cancer cells as well as its proliferation and survival. The expression of these mRNAs is increased in malignant tumors and strongly induced in different cancer cell lines by hypoxia inducible factor (HIF) through active HIF binding sites in promoter region of PFKFB-4 and PFKFB-3 genes. Moreover, the expression and hypoxia responsibility of PFKFB-4 and PFKFB-3 was also shown for pancreatic (Panc1, PSN-1, and MIA PaCa-2) as well as gastric (MKN45 and NUGC3) cancer cells. At the same time, their basal expression level and hypoxia responsiveness vary in the different cells studied: the highest level of PFKFB-4 protein expression was found in NUGC3 gastric cancer cell line and lowest in Panc1 cells, with a stronger response to hypoxia in the pancreatic cancer cell line. Overexpression of different PFKFB in pancreatic and gastric cancer cells under hypoxic condition is correlated with enhanced expression of vascular endothelial growth factor (VEGF) and Glut1 mRNA as well as with increased level of HIF-1&#x003b1; protein. Increased expression of different PFKFB genes was also demonstrated in gastric, lung, breast, and colon cancers as compared to corresponding non-malignant tissue counterparts from the same patients, being more robust in the breast and lung tumors. Moreover, induction of PFKFB-4 mRNA expression in the breast and lung cancers is stronger than PFKFB-3 mRNA. The levels of both PFKFB-4 and PFKFB-3 proteins in non-malignant gastric and colon tissues were more pronounced than in the non-malignant breast and lung tissues. It is interesting to note that Panc1 and PSN-1 cells transfected with dominant/negative PFKFB-3 (dnPFKFB-3) showed a lower level of endogenous PFKFB-3, PFKFB-4, and VEGF mRNA expressions as well as a decreased proliferation rate of these cells. Moreover, a similar effect had dnPFKFB-4. In conclusion, there is strong evidence that PFKFB-4 and PFKFB-3 isoenzymes are induced under hypoxia in pancreatic and other cancer cell lines, are overexpressed in gastric, colon, lung, and breast malignant tumors and undergo changes in their metabolism that contribute to the proliferation and survival of cancer cells. Thus, targeting these PFKFB may therefore present new therapeutic opportunities.