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.

Tribulus terrestris extracts alleviate muscle damage and promote anaerobic performance of trained male boxers and its mechanisms： Roles of androgen, IGF-1, and IGF binding protein-3

Purpose: To investigate the effects of Tribulus terrestris(TT) extracts on muscle mass, muscle damage, and anaerobic performances of trained male boxers and its mechanisms: roles of plasma androgen, insulin growth factor 1(IGF-1), and IGF-1 binding protein-3(IGFBP-3).Methods: Fifteen male boxers were divided into exercise group(E, n = 7) and exercise plus TT group(E + TT, n = 8). The 2 groups both undertook3-week high-intensity and 3-week high-volume trainings separated by a 4-week rest. TT extracts(1250 mg/day) were orally administered by boxers in E + TT group. TT extract compositions were detected by UHPLC–Q-TOF/MS. Before and at the end of the 2 trainings, muscle mass, anaerobic performance, and blood indicators were explored.Results: Compared with E group, decreases of plasma CK(1591.5 ± 909.6 U/L vs. 2719.9 ± 832.5 U/L) and IGFBP-3(3075.5 ± 1072.5 ng/m L vs. 3950.8 ± 479.3 ng/m L) as well as increases of mean power(MP, 459.4 ± 122.3 W vs. 434.6 ± 69.5 W) and MP/body weight(MP/BW, 7.5 ± 0.9 W/kg vs. 7.1 ± 1.1 W/kg) were detected in E + TT group after a high-intensity training. For high-volume training, reduction of IGFBP-3(2946.4 ± 974.1 ng/m L vs. 3632.7 ± 470.1 ng/m L) and increases of MP(508.7 ± 103.2 W vs. 477.8 ± 49.9 W) and MP/BW(8.2 ± 0.3 W/kg vs.7.5 ± 0.9 W/kg) were detected in E + TT group, compared with E group. Muscle mass, blood levels of testosterone, dihydrotestosterone(DHT),and IGF-1 were not signifiantly changed between the 2 groups.Conclusion: Taking 1250 mg capsules containing TT extracts did not change muscle mass and plasma levels of testosterone, DHT, and IGF-1 but significantly alleviated muscle damage and promoted anaerobic performance of trained male boxers, which may be related to the decrease of plasma IGFBP-3 rather than androgen in plasma.

Significance of highly phosphorylated insulin-like growth factor binding protein-1 and cervical length for prediction of preterm delivery in twin pregnancies

BACKGROUND A twin pregnancy can carry greater risks than singleton pregnancies.About 60 in 100 twin pregnancies result in spontaneous birth before 37 wk,which is associated with several complications in the premature babies.Clinical detection of biomarkers may help to predict the possibility of premature birth so that corresponding interventions can be given to the pregnant women in a timely manner,in order to reduce the risk of preterm birth and improve the outcomes of the newborn infants.AIM To explore the clinical value of transvaginal ultrasound measurement of cervical length combined with insulin-like growth factor binding protein-1(IGFBP-1)hyperphosphorylation in cervical secretions as predictors of preterm delivery in twin pregnancies.METHODS A total of 254 pregnant women with twin pregnancies,who were admitted to Hainan General Hospital and underwent maternity examination,were selected as the study subjects from January 2015 to December 2018.All participants received transvaginal ultrasound measurement of cervical length and phosphorylated IGFBP-1(phIGFBP-1)test between 24 and 34 wk gestation.The pregnancy outcomes were analyzed.RESULTS Of the women with a positive phIGFBP-1 test result,preterm birth rate was higher in those with a cervical length≤25 mm than those with a cervical length>25 mm(all P<0.05).Similarly,in women with a negative phIGFBP-1 test result,preterm birth rate was higher in those with a cervical length≤25 mm than those with a cervical length>25 mm(all P<0.05).The sensitivity,specificity,and positive and negative predictive values of the phIGFBP-1 test combined with the cervical length test were 95.71%,91.21%,95.12%and 92.22%,respectively,for the prediction of preterm birth.CONCLUSION Cervical length combined with phIGFBP-1 tests is of value for the prediction of outcomes of preterm delivery in twin pregnancies.

Exosomes derived from microglia overexpressing miR-124-3p alleviate neuronal endoplasmic reticulum stress damage after repetitive mild traumatic brain injury

We previously reported that miR-124-3p is markedly upregulated in microglia-derived exosomes following repetitive mild traumatic brain injury.However,its impact on neuronal endoplasmic reticulum stress following repetitive mild traumatic brain injury remains unclear.In this study,we first used an HT22 scratch injury model to mimic traumatic brain injury,then co-cultured the HT22 cells with BV2 microglia expressing high levels of miR-124-3p.We found that exosomes containing high levels of miR-124-3p attenuated apoptosis and endoplasmic reticulum stress.Furthermore,luciferase reporter assay analysis confirmed that miR-124-3p bound specifically to the endoplasmic reticulum stress-related protein IRE1α,while an IRE1αfunctional salvage experiment confirmed that miR-124-3p targeted IRE1αand reduced its expression,thereby inhibiting endoplasmic reticulum stress in injured neurons.Finally,we delivered microglia-derived exosomes containing miR-124-3p intranasally to a mouse model of repetitive mild traumatic brain injury and found that endoplasmic reticulum stress and apoptosis levels in hippocampal neurons were significantly reduced.These findings suggest that,after repetitive mild traumatic brain injury,miR-124-3 can be transferred from microglia-derived exosomes to injured neurons,where it exerts a neuroprotective effect by inhibiting endoplasmic reticulum stress.Therefore,microglia-derived exosomes containing miR-124-3p may represent a novel therapeutic strategy for repetitive mild traumatic brain injury.

Possible roles of insulin, IGF-1 and IGFBPs in initiation and progression of colorectal cancer

AIM: To investigate the roles of serum insulin, insulin-like growth factor-1 (IGF-1), and insulin-like growth factor binding proteins (IGFBPs) in the initiation and progression of colorectal cancer.

Effect of Dangfei Liganning capsule(当飞利肝宁胶囊) on liver X receptor α/steroid regulatory element binding protein-1/fatty acid synthase signal pathway in rats with metabolic-associated fatty liver disease

OBJECTIVE: To study the mechanism of Dangfei Liganning capsule(当飞利肝宁胶囊) in the treatment of rats with metabolic associated fatty liver disease(MAFLD). METHODS: Totally 48 specific pathogen free SpragueDawley male rats were randomly divided into normal Group, model group, Dangfei Liganning high, moderate, and low-dose groups and Essentiale group which were fed with high fat diet for 8 weeks, and gavage and molding were carried out simultaneously. Dangfei Liganning high, middle and low-dose group were given 0.27, 0.135 and 0.0675 g·kg-1·d-1 respectively by gavage, Essentiale group was given 0.123 g·kg-1·d-1 by gavage, the same amount of distilled water was given by gavage in the normal group and the model group. The rats were weighed at the 0th week, 2nd week, 4th week, 6th week and 8th weekend respectively. The rats were sacrificed at the end of the 8th week. Serum levels of alanine aminotransferase(ALT), alanine aminotransferase(AST),triglyceride(TG), total cholesterol(CHO), high-density lipoprotein cholesterol(HDL-C), low-density lipoprotein (LDL-C), total protein(TP), albumin(Alb), globulin(GLB), total bilirubin(TBIL), direct bilirubin(DBIL), tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6) were measured. The levels of liver tumor necrosis factor-α(TNF-α), interleukin-6(IL-6) and liver pathology [hematoxylin and eosin(HE) staining, oil red O staining] were detected. The expression levels of liver X receptor α(LXRα), steroid regulatory element binding protein-1(SREBP-1) and fatty acid synthase(FAS) were detected by immunohistochemistry, Western blot and reverse transcription-polymerase chain reaction reverse transcription-polymerase chain reaction. RESULTS: From the beginning to the 8th week, the growth rate of body weight in the Dangfei Liganning highdose group was slower than all other groups. There was no significant difference in ALB level in all groups(P > 0.05). Compared with the model group, the levels of ALT, AST, LDL-C, TG, CHO, TP, GLB, TBIL, DBIL, IL-6, TNF-α were significantly decreased and HDL-C were significantly increased in Dangfei Liganning high-dose group(P < 0.01, < 0.05). HE and oil red O staining showed that the fatty lesions in rat liver were alleviated, while the expressions of LXRα, SREBP-1, FAS m RNA and protein were significantly decreased(P < 0.01). CONCLUSIONS: Dangfei Liganning capsule can slow down the increase of body weight of MAFLD rats, reduce the levels of transaminase, Lipid and inflammatory factors in MAFLD rats, promote the synthesis of liver protein and bile metabolism, and improve the liver fatty lesion of MAFLD rats, among which the Dangfei Liganning highdose group is more effective. The mechanism of action may be through blocking LXR-SREBP-1-FAS signal pathway.

Combination of insulin-like growth factor-1, IGF binding protein-3, chromogranin A and prostate specific antigen can improve the detection of prostate cancer

Aim:Prostate cancer(PCa)is the second most prevalent male cancer worldwide and designated the sixth most frequent male cancer in Arab countries.Although prostate specific antigen(PSA)has become the best and most valuable biomarker for screening of PCa,elevated levels of PSA can reflect the presence of malignant cells but can overlap with benign prostatic diseases.There is a necessity to develop and improve current tools for early detection and diagnosis of PCa.This study was done to evaluate the validation of serum insulin-like growth factor-1(IGF-1),IGF binding protein-3(IGFBP-3),chromogranin A(CgA)and combination with PSA in treatment of benign prostatic hyperplasia(BPH)and PCa patients.Methods:The study included 72 patients with PCa,70 BPH patients and 56 healthy male subjects of matched age.Full history and clinical data were recorded for all subjects.Results:Serum PSA attained sensitivity of 84%at 82%specificity with an accuracy of 83%,although IGF-1,IGFBP-3 and CgA did not recognize PCa patients.Conclusion:Combinations of IGF-1 and IGFBP-3 biomarkers with PSA were effectively differentiated between PCa and control groups as well as improving the overall value of sensitivity,specificity and diagnostic accuracy of PCa to 85%and 86%for IGF-1/PSA and IGFP-3/PSA respectively.

Effect of TRAF6 gene silencing on hepatocellular carcinoma cell line SMCC7721 and its possible mechanism

Objective:To explore the effect of TRAF6 gene silencing on the function of hepatocellular carcinoma SMCC7721 and its possible mechanism.Method:Cell lines were constructed by cell transfection technology and verified by quantitative real-time PCR.Cell functional changes were observed by CCK8 method,Transwell test and Method of EdU.Western blotting was used to explore the possible mechanism of action.Result:TRAF6 RNA was abnormally up-regulated in HCC,and TRAF6 levels were detected in both HCC cell lines and L02 cells.SMCC7721 was selected as TRAF6 high expression cell.The results of CCK8 assay and EdU method showed that the decrease of TRAF6 expression significantly inhibited the proliferation of SMCC7721 cells.The results of CCK8 assay and EdU method showed that the decrease of TRAF6 expression significantly inhibited the proliferation of SMCC7721 cells.Overexpression of TRAF6 in TRAF6 knockdown cells can restore and enhance cell proliferation.Transwell assay confirmed that the invasiveness of SMCC 7721 cells treated with siRNA was significantly reduced.After treatment with LV-Rescue plasmid,the cell invasion was restored and enhanced.Western blotting showed that the protein levels of YB-1,Wnt,β-catenin,c-myc and Cyclin D1 were significantly down-regulated in siRNA group.On the contrary,the expression level of CYLD protein increased.Conclusion:As an important intracellular junctive protein in tumor cells,TRAF6 may improve the expression of pro-cancer factors C-myc and Cyclin D1 by modifying(ubiquitination)YB-1,thus improving the proliferation ability of cells.This process may be closely positively correlated with the Wnt/β-catenin pathway,and negatively correlated with the expression of CYLD protein.

Effects of endoplasmic reticulum stress on the expressionof inflammatory cytokines in patients with ulcerative colitis

AIM: To explore the changes of X-box binding protein 1splicing(XBP1s) and inflammatory cytokine expression in patients with ulcerative colitis(UC) in response to endoplasmic reticulum stress(ERS).METHODS: Reverse transcription polymerase chain reaction and quantitative polymerase chain reaction were performed to detect the forms of XBP1 s and the expression of interleukin(IL)-2, interferon(IFN)-γ, and IL-17α. Differences between patients with UC and normal subjects were then determined.RESULTS: Mononuclear cells of the peripheral blood of normal subjects and UC patients with were stimulated with no drugs(control), phytohemagglutinin(PHA), thapsigargin(TG), or both PHA and TG. XBP1 s in patients with UC exhibited splicing, which was greater with co-stimulation than single stimulation. Costimulation increased the expression level of IL-2, IFN-γ, and IL-17α.CONCLUSION: The T lymphocytes of both normal subjects and patients with UC responded to ERS by activating the XBP1s-mediated signalling pathway, upregulating the expression of inflammatory cytokines, and increasing the occurrence of inflammation. The mononuclear cells in the peripheral blood of patients with UC were more sensitive to ERS than those in the peripheral blood of normal subjects.

Resveratrol and fenofibrate ameliorate fructose-induced nonalcoholic steatohepatitis by modulation of genes expression

AIM: To evaluate the effect of resveratrol, alone and in combination with fenofibrate, on fructose-induced metabolic genes abnormalities in rats.METHODS: Giving a fructose-enriched diet (FED) to rats for 12 wk was used as a model for inducing hepatic dyslipidemia and insulin resistance. Adult male albino rats (150-200 g) were divided into a control group and a FED group which was subdivided into 4 groups, a control FED, fenofibrate (FENO) (100 mg/kg), resveratrol (RES) (70 mg/kg) and combined treatment (FENO + RES) (half the doses). All treatments were given orally from the 9th week till the end of experimental period. Body weight, oral glucose tolerance test (OGTT), liver index, glucose, insulin, insulin resistance (HOMA), serum and liver triglycerides (TGs), oxidative stress (liver MDA, GSH and SOD), serum AST, ALT, AST/ALT ratio and tumor necrosis factor-α (TNF-α) were measured. Additionally, hepatic gene expression of suppressor of cytokine signaling-3 (SOCS-3), sterol regulatory element binding protein-1c (SREBP-1c), fatty acid synthase (FAS), malonyl CoA decarboxylase (MCD), transforming growth factor-β1 (TGF-β1) and adipose tissue genes expression of leptin and adiponectin were investigated. Liver sections were taken for histopathological examination and steatosis area were determined.RESULTS: Rats fed FED showed damaged liver, impairment of glucose tolerance, insulin resistance, oxidative stress and dyslipidemia. As for gene expression, there was a change in favor of dyslipidemia and nonalcoholic steatohepatitis (NASH) development. All treatment regimens showed some benefit in reversing the described deviations. Fructose caused deterioration in hepatic gene expression of SOCS-3, SREBP-1c, FAS, MDA and TGF-β1 and in adipose tissue gene expression of leptin and adiponectin. Fructose showed also an increase in body weight, insulin resistance (OGTT, HOMA), serum and liver TGs, hepatic MDA, serum AST, AST/ALT ratio and TNF-α compared to control. All treatments improved SOCS-3, FAS, MCD, TGF-β1 and leptin genes expression while only RES and FENO + RES groups showed an improvement in SREBP-1c expression. Adiponectin gene expression was improved only by RES. A decrease in body weight, HOMA, liver TGs, AST/ALT ratio and TNF-α were observed in all treatment groups. Liver index was increased in FENO and FENO + RES groups. Serum TGs was improved only by FENO treatment. Liver MDA was improved by RES and FENO + RES treatments. FENO + RES group showed an increase in liver GSH content.CONCLUSION: When resveratrol was given with half the dose of fenofibrate it improved NASH-related fructose-induced disturbances in gene expression similar to a full dose of fenofibrate.

The unfolded protein response signaling and retinal Müller cell metabolism

The retina is one of the most energy demanding tissues in the body. Like most neurons in the central nervous system, retinal neurons consume high amounts of adenosine-5′-triphosphate（ATP） to generate visual signal and transmit the information to the brain. Disruptions in retinal metabolism can cause neuronal dysfunction and degeneration resulting in severe visual impairment and even blindness. The homeostasis of retinal metabolism is tightly controlled by multiple signaling pathways, such as the unfolded protein response（UPR）, and the close interactions between retinal neurons and other retinal cell types including vascular cells and Müller glia. The UPR is a highly conserved adaptive cellular response and can be triggered by many physiological stressors and pathophysiological conditions. Activation of the UPR leads to changes in glycolytic rate, ATP production, de novo serine synthesis, and the hexosamine biosynthetic pathway, which are considered critical components of Müller glia metabolism and provide metabolic support to surrounding neurons. When these pathways are disrupted, neurodegeneration occurs rapidly. In this review, we summarize recent advance in studies of the UPR in Müller glia and highlight the potential role of the UPR in retinal degeneration through regulation of Müller glia metabolism.

Holistic anti-tumor resistance mechanism of YBX1 and its potential as a chemoresistance target in pancreatic ductal adenocarcinoma

The overall survival rate of pancreatic ductal adenocarcinoma(PDAC)is the worst among all cancers,which is mainly due to the fact that most patients are in the late tumor stage when diagnosed,lacking effective treatment options.Although targeted therapy has shown some prospects in PDAC,its efficacy is limited to patients with specific gene mutation or target gene expression.A large number of patients have no other treatment options except chemotherapy.However,the high drug resistance rate of chemotherapy for PDAC severely limits the improvement of curative effect.Therefore,determining the key factors that lead to drug resistance in PDAC is crucial to improve the prognosis of patients.Multifunctional oncoprotein Y-box binding protein 1(YBX1)may be one of such potential targets.Studies have confirmed that YBX1 is associated with the inherent behavior of a variety of cancers,such as proliferation,invasion,metastasis,and cancer cell stemness.Herein,we integrated and analyzed the resistance mechanism of YBX1 in anti-tumor therapy,and discussed its potential as a therapeutic target to reverse the chemotherapy resistance of PDAC.

过表达特异性核基质蛋白1基因对裸鼠前列腺癌LNCaP细胞移植瘤生长的影响及其机制

目的 观察过表达特异性核基质蛋白1（SATB1）基因对裸鼠前列腺癌LNCaP细胞移植瘤生长的影响并探讨其作用机制.方法 利用LipofectamineTM 2000将pcDNA3.1-SATB1、pcDNA3.1转染至人前列腺癌LNCaP细胞,建立前列腺癌LNCaP荷瘤鼠模型,分为3组：转染pcDNA3.1-SATB1LNCaP组、转染空质粒pcDNA3.1LNCaP组和未转染LNCaP细胞组,每组各8只.取浓度为2＆#215;1010/L转染pcDNA3.1-SATB1的DU-145、转染空质粒pcDNA3.1的DU-145和未转染的DU-145细胞悬液0.2ml分别注射至各组裸鼠左腋皮下.每隔4d测量皮下移植瘤体积,绘制瘤体生长曲线.免疫组织化学法和Western blot法检测各组瘤体SATB1的表达.免疫组织化学法检测各组瘤体E-钙黏蛋白（E-cadherin）、波形蛋白（Vimentin）、基质金属蛋白酶（MMP）-2的表达.原位缺口末端标记法（TUNEL）法检测移植瘤凋亡.结果 Western blot表明：转染pcDNA3.1-SATB1LNCaP组可过表达SATB1基因并成功构建人前列腺癌LNCaP细胞裸鼠皮下移植瘤模型.第27天时处死裸鼠后,转染pcDNA3.1-SATB1LNCaP组移植瘤体积为（2242.0±259.2） mm3,显著高于对照组,差异有统计学意义（P ＜0.05）;TUNEL结果显示：转染pcDNA3.1-SATB1LNCaP组平均凋亡率为（31.3±8.9）％,与对照组比较,差异有统计学意义（P＜0.05）;免疫组织化学法显示：Vimentin、MMP-2、E-cadherin在转染pcDNA3.1-SATB1 LNCaP组的表达分别为417.9±18.2、539.1±41.6和156.4±11.9,与对照组比较,蛋白表达差异均有统计学意义（P＜0.05）.结论 成功构建过表达SATB1基因的前列腺癌LNCaP细胞荷瘤鼠模型.SATB1可促进前列腺癌细胞的增殖生长、抑制其凋亡.SATB1可通过调控E-cadherin、Vimentin、MMP-2的表达水平,进而参与调控前列腺癌的侵袭和转移.

未折叠蛋白反应相关因子葡萄糖调节蛋白78、X-盒结合蛋白1在食管癌中的表达及其意义

目的：探讨未折叠蛋白反应相关因子葡萄糖调节蛋白78（GRP78）及X-盒结合蛋白1（XBP1）在食管鳞状细胞癌中的表达、其对内质网应激（ERS）的激活作用及其在食管癌浸润生长中的作用机制。方法通过免疫组织化学方法检测30例食管鳞状细胞癌及30例正常食管鳞状上皮组织中GRP78、XBP1的表达，分析其与患者预后的关系。结果食管鳞状细胞癌中GRP78阳性表达率为83.3%（25／30），食管正常鳞状上皮组织中为20.0%（6／30）（χ2＝25.833，P＜0.05）。食管鳞状细胞癌中XBP1阳性表达率为70.0%（21／30），食管正常鳞状上皮组织中为26.7%（8／30）（χ2＝20.872，P＜0.05）。肿瘤浸润肌层者的GRP78和XBP1阳性表达率明显高于浸润黏膜层者。结论食管鳞状细胞癌组织中GRP78和XBP1高表达，可能参与了食管鳞状细胞癌的发生、发展。