Synaptotagmins family affect glucose transport in retinal pigment epithelial cells through their ubiquitination-mediated degradation and glucose transporter-1 regulation

BACKGROUND Synaptotagmins(SYTs)are a family of 17 membrane transporters that function as calcium ion sensors during the release of Ca2+-dependent neurotransmitters and hormones.However,few studies have reported whether members of the SYT family play a role in glucose uptake in diabetic retinopathy(DR)through Ca2+/glucose transporter-1(GLUT1)and the possible regulatory mechanism of SYTs.AIM To elucidate the role of the SYT family in the regulation of glucose transport in retinal pigment epithelial cells and explore its potential as a therapeutic target for the clinical management of DR.METHODS DR was induced by streptozotocin in C57BL/6J mice and by high glucose medium in human retinal pigment epithelial cells(ARPE-19).Bioinformatics analysis,reverse transcriptase-polymerase chain reaction,Western blot,flow cytometry,ELISA,HE staining,and TUNEL staining were used for analysis.RESULTS Six differentially expressed proteins(SYT2,SYT3,SYT4,SYT7,SYT11,and SYT13)were found between the DR and control groups,and SYT4 was highly expressed.Hyperglycemia induces SYT4 overexpression,manipulates Ca2+influx to induce GLUT1 fusion with the plasma membrane,promotes abnormal expression of the glucose transporter GLUT1 and excessive glucose uptake,induces ARPE-19 cell apoptosis,and promotes DR progression.Parkin deficiency inhibits the proteasomal degradation of SYT4 in DR,resulting in SYT4 accumulation and enhanced GLUT1 fusion with the plasma membrane,and these effects were blocked by oe-Parkin treatment.Moreover,dysregulation of the myelin transcription factor 1(Myt1)-induced transcription of SYT4 in DR further activated the SYT4-mediated stimulus-secretion coupling process,and this process was inhibited in the oe-MYT1-treated group.CONCLUSION Our study reveals the key role of SYT4 in regulating glucose transport in retinal pigment epithelial cells during the pathogenesis of DR and the underlying mechanism and suggests potential therapeutic targets for clinical DR.

Effects of suppressing glucose transporter-1 by an antisense oligodeoxynucleotide on the growth of human hepatocellular carcinoma cells

BACKGROUND:The glucose transporter-1(Glut-1),a key ratelimiting factor in the transport and metabolism of glucose in cancer cells,is over-expressed in many human cancer cells and this overexpression is correlated with poor biological behavior. The increased levels of Glut-1 expression in hepatocellular carcinoma(HCC)cells functionally affect tumorigenicity.This study was undertaken to investigate effects of suppressing Glut-1 by an antisense oligodeoxynucleotide(AS-ODN)on the growth of human hepatocellular carcinoma(HepG-2)cells. METHODS:We used AS-ODN targeting against the Glut-1 gene in a HepG-2 cell line.There were four experimental groups: empty pcDNA3.1 vector(mock transfection),pcDNA3.1-anti-Glut(+),pcDNA3.1-Glut(+),and non-transfected HepG-2 cells. The Glut-1 mRNA expression was detected by RT-PCR and the Glut-1 protein expression by Western blotting after cell culture, and the glucose uptake was detected after glucose stimulation in each group. RESULTS:Compared with non-transfected HepG-2 or Glut-1 pcDNA3.1,a down-regulation of Glut-1 mRNA in HepG-2 cells transfected with anti-Glut-1 pcDNA3.1 was noted(P<0.05).Glut-1 protein in HepG-2 cells transfected with Glut-1 AS-ODN was decreased compared with non-transfected HepG-2,Glut-1 pcDNA3.1,or empty vectors. Glucose uptake by the HepG-2 cells transfected with AS-ODN was decreased at 1 hour after glucose stimulation.CONCLUSIONS:The application of Glut-1 AS-ODN can down-regulate the expression of Glut-1 at mRNA and protein,and inhibit glucose uptake partially in HepG-2 cells.The Glut-1 gene maybe a potential therapeutic target for HCC.

Proprotein convertase 1/3-mediated down-regulation of brain-derived neurotrophic factor in cortical neurons induced by oxygen-glucose deprivation

Brain-derived neurotrophic factor(BDNF)has robust effects on synaptogenesis,neuronal differentiation and synaptic transmission and plasticity.The maturation of BDNF is a complex process.Proprotein convertase 1/3(PC1/3)has a key role in the cleavage of protein precursors that are directed to regulated secretory pathways;however,it is not clear whether PC1/3 mediates the change in BDNF levels caused by ischemia.To clarify the role of PC1/3 in BDNF maturation in ischemic cortical neurons,primary cortical neurons from fetal rats were cultured in a humidified environment of 95%N_2 and 5%CO_2 in a glucose-free Dulbecco's modified Eagle's medium at 37℃for3 hours.Enzyme-linked immunosorbent assays and western blotting showed that after oxygen-glucose deprivation,the secreted and intracellular levels of BDNF were significantly reduced and the intracellular level of PC1/3 was decreased.Transient transfection of cortical neurons with a PC1/3 overexpression plasmid followed by oxygen-glucose deprivation resulted in increased PC1/3 levels and increased BDNF levels.When levels of the BDNF precursor protein were reduced,the concentration of BDNF in the culture medium was increased.These results indicate that PC 1/3 cleavage of BDNF is critical for the conversion of pro-BDNF in rat cortical neurons during ischemia.The study was approved by the Animal Ethics Committee of Wuhan University School of Basic Medical Sciences.

IDENTIFICATION OF GLUCOSE TRANSPORTER- 1 AND ITS FUNCTIONAL ASSAY IN MOUSE GLOMERULAR MESANGIAL CELLS CULTURED IN VITRO

To evaluate the role of glucose transporter- l (GLUT1) in the glucose uptake of glomerular mesangial cells. Methods. Cultured C57/SJL mouse mesangial cells were used in the study. The expression of GLUT1 mRNA was detected by RT- PCR. The expression of GLUT1 protein was detected by immunofluorescence and flow cytometry. The uptake of glucose and its kinetics were determined by 2- deoxy- [3H]- D- glucose uptake. Results. Both GLUT1 mRNA and protein were found in mouse glomerular mesangial cells. 2- deoxy- D- glucose uptake and kinetics assay showed that this glucose transporter had high affinity for glucose and the glucose uptake specificity was further confirmed by phloretin. Conclusion. Functional GLUT1 did present in mouse mesangial cells cultured in vitro and it might be the predominant transporter mediated the uptake of glucose into mesangial cells.

Constructing recombinant replication-defective adenoviral vectors that express glucose transporter-1 through in vitro ligation

BACKGROUND： We constructed a homologous recombination bacterial method based on the pAdEasy system, a widely used system, for generating recombinant adenoviral vectors that express glucose transporter- 1 （GLUT 1） in rats, OBJECTIVE： This study was designed to investigate the feasibility of generating recombinant replication-defective adenoviral vectors that express GLUT1 in rats by in vitro ligation based on the Adeno-X^TM system. DESIGN： An in vitro cell-based experiment. SETTING： This study was performed at the Linbaixin Medical Research Center of the Second Hospital Affiliated to Sun Yat-sen University and Central Laboratory for Prevention and Treatment of Tumor, Sun Yat-sen University between January and August 2004. MATERIALS： Male, adult, Sprague Dawley rats were used to extract total RNA from brain tissue. E. coli DH5 a and human embryonic kidney 293 cells （HEK293 cells） used in the present study were cryo-preserved by the Second Hospital Affiliated to Sun Yat-sen University. Rabbit anti-rat GLUT1 polyclonal antibody （Chemicon, U.S.A.） and primers （Shanghai Boya Bioengineering Co., Ltd） were also used. METHODS： E1/E3-deleted replication-defective adenoviral vectors were used. Using in vitro ligation, the target gene was first sub-cloned into a shuttle vector plasmid to obtain the fragment containing target gene expression cassettes by enzyme digestion. Subsequently, the fragment was co-transformed with linearized adenoviral backbone vector into the E. coli strain. The recombinant adenoviral plasmid was transfected into HEK293 cells to assembly recombinant adenoviral vectors with replication capabilities. The procedure was repeated several times for recombinant adenoviral vectors amplification. MAIN OUTCOME MEASURES： Efficiency of recombinant adenoviral vectors to express the target gene was measured by gene and protein expression through polymerase chain reaction and Western Blot assays, respectively. RESULTS： Results demonstrated that recombinant adenoviral vectors successfully expressed GLUT1 protein, with a relative molecular mass of 55000 in HEK293 cells. These results suggest that recombinant adenoviral vectors obtained by homologous bacterial recombination feature high efficiency, rapidness, and simplicity. CONCLUSION： We successfully amplified the rat GLUT1 gene and constructed replication-defective adenoviral vectors expressing GLUT1. The replication-defective adenoviral vectors proved to successfully express the target gene in HEK293 cells.

Lower Concentrations of Glucose or Insulin Decrease the Risk of Various Types of Cancer in the Long-Lived Ames Dwarf Mouse by Increasing the Expression of p27Kip1, a Cell-Cycle Repressor Protein

Introduction. The molecular biological mechanism of the increased incidence of the various types of cancer in obesity or type 2 diabetes in rodents or humans has largely been resolved in recent years. By contrast, the molecular biological mechanism of the decreased, not increased, incidence of the various types of cancer in the homozygous long-lived Ames dwarf mice still remains unresolved. Objective. The first objective of the present study was to investigate whether the decrease in the incidence of cancer in the homozygous long-lived Ames dwarf mice is due to the increase, not decrease, in the expression of p27Kip1, a cell cycle repressor protein. The second objective was to investigate whether the decrease in the incidence of cancer in the homozygous long-lived Ames dwarf mice is due to the decrease, not increase, in the levels of glucose or insulin. Methods. To achieve these objectives, we first performed western immunoblot analysis of the hepatic expression of p27Kip1 protein. We then performed, using a human breast cancer cell line in vitro, the luciferase reporter plasmid assay to determine whether the translation initiation activity of the p27Kip1 mRNA is increased when the concentrations of either glucose or insulin are decreased. Results and Conclusion. The results of the first objective indicated that the hepatic expression of p27Kip1 protein was up-regulated in the homozygous long-lived Ames dwarf mice as expected. We also found that the lower concentrations of glucose or insulin increased the translation initiation activity of the p27Kip1 mRNA.

High Glucose Promotes the CTGF Expression in Human Mesangial Cells via Serum and Glucocorticoid-induced Kinase 1 Pathway

The role of serum and glucocorticoid-induced kinase 1 （SGK1） pathway in the connective tissue growth factor （CTGF） expression was investigated in cultured human mesangial cells （HMCs） under high glucose. By using RT-PCR and Western blot, the effect of SGK1 on the CTGF expression in HMCs under high glucose was examined. Overexpression of active SGK1 in HMCs transfected with PIRES2-EGFP- S422D hSGK1 （SD） could increase the expression of phosphorylated SGK1 and CTGF as compared with HMCs groups transfected with PIRES2-EGFP （FP） under high glucose or normal glucose. Overexpression of inactive SGK1 in HMCs transfected with PIRES2-EGFP- K127N hSGK1 （KN） could decrease phosphorylated SGK1 and CTGF expression as compared with HMCs groups transfected with FP under high glucose. In conclusion, these results suggest that high glucose-induced CTGF expression is mediated through the active SGK1 in HMCs.

Neuroprotection of quercetin on central neurons against chronic high glucose through enhancement of Nrf2/Glo-1 mediated by phosphorylation regulation

OBJECTIVE To investigate the neuroprotective effects of quercetin on central neurons against chronic high glucose in central neurons,in relation to Nrf2/ARE/Glo-1 activation.METHODS SH-SY5Y cells were cultured with high glucose(HG,70 mmol·L^(-1)),4-fold of the normal glucose(17.5 mmol·L^(-1)).Quercetin was set three concentrations(5,10,20μmol·L^(-1)),with Nrf2 activator sulforaphane(SFN)as a positive group(2.5μmol·L^(-1)).After 72 h,cells were collected for glyoxalase 1(Glo-1)activity and GSH level were by spectrophotometry;advanced glycation end-products(AGEs)as well as nuclear Nrf2 and p-Nrf2 levels by immunofluorescence;Glo-1,γ-glutamycysteine synthase(γ-GCS),Nrf2 and p-Nrf2 protein levels by Western blotting,and Glo-1 andγ-GCS m RNA levels by real-time qP CR.RESULTS Quercetin increased the cell viability of SH-SY5Y cells,and upregulated the levels of Glo-1 activity,protein,and m RNA in SH-SY5Y cells cultured with HG,accompanied by the elevated levels of glutathione,a cofactor of Glo-1 activity,and the reduced levels of AGEs.Meanwhile,quercetin could increase p-Nrf2 and Nrf2 levels in nucleus as well as p-Nrf2 levels in cytosol of SH-SY5Y cells exposed to chronic HG,accompanied by the elevated protein expression and m RNA levels ofγ-GCS,a known target gene of Nrf2/ARE signaling.Moreover,a PKC activator or a p38MAPK inhibitor pretreatment could significantly increase the protein expression ofγ-GCS in HG condition,but an alkylating agent for sulfydryl of cysteine in Keap 1,a negative regulator of Nrf2,pretreatment only showed an increased tendency ofγ-GCS protein,compared with without pretreatment;however,after pretreatment with those tool drugs,co-treatment with quercetin and HG had similar results to those of single tool drug pretreatment followed by HG exposure.CONCLUSION Firstly,quercetin can enhance Glo-1 function in central neurons,which is mediated by activation of Nrf2/ARE pathway,then exerts the neuroprotection against HG induced damage;moreover,PKC and p38 MAPK pathways may be involved in Nrf2 inactivation in chronic HG condition.

Host Factors Alter Effects of Angiopoietin-Like Protein 8 on Glucose Homeostasis in Diabetic Mice

Recovery of functional beta cell mass offers a biological cure for type 1 diabetes. However, beta cell mass is difficult to regain once lost since the proliferation rate of beta cells after youth is very low. Angiopoietin like-protein 8 (ANGPTL8), a peptide that has a role in the regulation of lipoprotein lipase activity, was reported to increase beta cell proliferation in mice in 2013. Subsequent studies of human ANGPTL8 for short term (3 to 8 days) in non-diabetic mice showed little or no increase in beta cell proliferation. Here, we examined the effect of ANGPTL8 on glucose homeostasis in models that have not been examined previously. We expressed mouse ANGPTL8 using adenovirus in 2 mouse models of diabetes (streptozotocin and Non-Obese Diabetic (NOD) mice) over 2 weeks. Also, we tested ANGPTL8 in NOD mice deficient in leukocyte 12-lipoxygenase (12LO), an enzyme that contributes to insulitis and loss of beta cell function in NOD, in an effort to determine whether 12LO deficiency alters the response to ANGPTL8. Adenovirus-mediated expression of ANGPTL8 lowered blood glucose levels in streptozotocin treated mice without an increase in beta cell proliferation or serum insulin concentration. While ANGPTL8 did not reverse hyperglycemia in overtly hyperglycemic NOD mice or alter glucose homeostasis of non-diabetic NOD mice, ANGPTL8 reduced blood glucose levels in 12LOKO NOD mice. However, the lower glucose levels in 12LOKO NOD were not associated with higher serum insulin levels or beta cell proliferation. In summary, while mouse ANGPTL8 does not increase beta cell proliferation in NOD mice or streptozotocin treated mice in agreement with studies in non-diabetic mice, it lowers blood glucose levels in multiple low-dose streptozotocin induced diabetes and 12LO deficiency indicating that host factors influence the impact of ANGPTL8 on glucose homeostasis.

高糖通过调控miR-429/ZEB1轴对胰腺癌细胞免疫逃逸的影响

目的探究高糖干预对胰腺癌细胞免疫逃逸的影响及作用分子机制。方法采用不同浓度葡萄糖(0、7.5、15、30 mmol/L)处理PANC-1细胞24 h构建高糖干预的PANC-1细胞。将miR-429 mimics及其阴性对照(mimics NC)转染至PANC-1细胞,分为对照组、HG组、HG+mimics NC组、HG+mimics组、HG+mimics+oe-NC组和HG+mimics+oe-ZEB1组。流式细胞术检测细胞表面分子细胞程序性死亡-配体1(PD-L1)表达水平;qRT-PCR检测细胞miR-429和锌指E-盒结合同源盒蛋白1(ZEB1)mRNA表达水平;Western blot检测细胞ZEB1蛋白表达水平。将以上各组PANC-1细胞与CD8^(+)T细胞建立共培养体系,CCK-8检测细胞增殖活性;流式细胞术检测细胞凋亡水平;乳酸脱氢酶(LDH)释放法检测CD8^(+)T细胞对PANC-1细胞的杀伤作用;采用双荧光素酶报告系统验证miR-429和ZEB1的靶向调控关系。结果HG可促进PANC-1细胞表面分子PD-L1及ZEB1表达(P<0.05),抑制miR-429表达,且呈浓度依赖性。miR-429过表达可显著抑制HG诱导的PANC-1细胞表面分子PD-L1表达,而过表达ZEB1可逆转miR-429过表达对HG诱导PANC-1细胞表面分子PD-L1表达的抑制作用。建立与CD8^(+)T细胞共培养体系后,与对照组比较,HG组PANC-1细胞增殖活性明显增加,细胞凋亡率和杀伤活性明显降低(P<0.05);与HG+mimics NC组比较,HG+mimics组PANC-1细胞增殖活性明显降低,细胞凋亡水平和杀伤活性明显升高(P<0.05)。与HG+mimics+oe-NC组比较,HG+mimics+oe-ZEB1组PANC-1细胞增殖活性明显增加,细胞凋亡率和杀伤活性明显降低(P<0.05)。双荧光素酶报告基因实验证实,miR-429靶向负调控ZEB1。结论高糖通过下调miR-429表达水平,靶向负调控ZEB1 mRNA的表达,提高PANC-1细胞表面分子PD-L1表达水平,进而促进PANC-1细胞免疫逃逸。

血清TyG指数和nesfatin-1及RBP4联合预测糖尿病视网膜病变的价值

目的:探究血清甘油三酯-葡萄糖(TyG)指数、摄食抑制因子-1(nesfatin-1)、视黄醇结合蛋白4(RBP4)联合预测糖尿病视网膜病变(DR)的价值,为DR早期预测提供支持。方法:回顾性分析。收集2022-02/2023-12我院接诊的2型糖尿病(T2DM)患者164例的临床资料,按照眼底检查结果分为DR组43例(其中增殖性DR 19例,非增殖性DR 24例),不合并DR的T2DM组121例。入院后记录患者基本资料,检查血清TyG指数、nesfatin-1、RBP4水平。结果:DR组病程长于T2DM组,空腹血糖、糖化血红蛋白、甘油三酯、总胆固醇、低密度脂蛋白及TyG指数、RBP4水平高于T2DM组,高密度脂蛋白、nesfatin-1水平低于T2DM组(均P<0.001)。多因素Logistic回归分析可知,T2DM病程(OR=1.338,95%CI:1.059-1.690)、糖化血红蛋白(OR=5.065,95%CI:1.659-15.470)、低密度脂蛋白(OR=12.715,95%CI:2.385-67.790)、TyG指数(OR=23.057,95%CI:2.936-181.073)、RBP4(OR=1.319,95%CI:1.028-1.692)是T2DM患者发生DR的危险因素,nesfatin-1(OR=0.007,95%CI:0.003-0.016)为保护因素。绘制ROC曲线显示,TyG指数、nesfatin-1、RBP4均对T2DM患者并发DR具有一定预测价值,曲线下面积(areas under curve,AUC)分别为0.804、0.878、0.738,各指标联合预测时AUC为0.946,预测敏感度为83.72%、特异度为92.56%。增殖性DR患者TyG指数、RBP4水平高于非增殖性DR患者,nesfatin-1水平低于非增殖性DR患者(均P<0.05)。Spearman相关性分析显示,TyG指数、RBP4水平与DR病情程度呈正相关,nesfatin-1水平与DR病情程度呈负相关(r_(s)=0.557、0.392、-0.359,均P<0.05)。Pearson相关分析显示,T2DM并发DR患者TyG指数与nesfatin-1水平呈负相关,与RBP4水平呈正相关,nesfatin-1与RBP4水平呈负相关(r=-0.486、0.538、-0.592,均P<0.05)。结论:血清TyG指数、nesfatin-1、RBP4水平与DR发病风险及病情程度有关,可作为DR早期预测的标志物,且联合预测效能更好。

瑞马唑仑调节HIF-1α/BNIP3信号通路对OGD/R诱导神经细胞自噬和凋亡的影响

目的探讨瑞马唑仑对OGD/R诱导的神经细胞自噬和凋亡的影响及作用机制。方法体外培养小鼠海马神经元细胞(HT22)并进行神经细胞氧糖剥夺/再复氧(OGD/R),筛选实验用瑞马唑仑浓度;将HT22细胞分为对照组、OGD/R组、瑞马唑仑组、2-ME2组、瑞马唑仑+2-ME2组;CCK8法检测5组HT22细胞活力;流式细胞术检测5组HT22细胞凋亡率;透射电子显微镜观察5组HT22细胞自噬小体的形成;Western blot检测5组HT22细胞HIF-1α、BNIP3、LC3-Ⅱ/LC3-Ⅰ的表达。结果确定实验用瑞马唑仑浓度为50μg/mL;与对照组比较,OGD/R组HT22细胞OD450值、HIF-1α、BNIP3、LC3-Ⅱ/LC3-Ⅰ蛋白水平下调,凋亡率上调(P<0.05);与OGD/R组比较,瑞马唑仑组HT22细胞自噬小体增加,OD450值、HIF-1α、BNIP3、LC3-Ⅱ/LC3-Ⅰ蛋白水平上调,凋亡率下调(P<0.05);2-ME2组HT22细胞OD450值、HIF-1α、BNIP3、LC3-Ⅱ/LC3-Ⅰ蛋白水平下调,凋亡率上调(P<0.05)。与瑞马唑仑组比较,瑞马唑仑+2-ME2组HT22细胞自噬小体数量减少,OD450值、HIF-1α、BNIP3、LC3-Ⅱ/LC3-Ⅰ蛋白水平下调,凋亡率上调(P<0.05);与2-ME2组比较,瑞马唑仑+2-ME2组HT22细胞OD450值、HIF-1α、BNIP3、LC3-Ⅱ/LC3-Ⅰ蛋白水平上调,凋亡率下调(P<0.05)。结论瑞马唑仑可通过激活HIF-1α/BNIP3信号通路促进OGD/R诱导的神经细胞自噬,抑制细胞凋亡,从而减轻OGD/R诱导的神经细胞损伤。

Overexpression of mitogen-activated protein kinase phosphatase-1 in endothelial cells reduces blood-brain barrier injury in a mouse model of ischemic stroke

Ischemic stroke can cause blood-brain barrier(BBB)injury,which worsens brain damage induced by stroke.Abnormal expression of tight junction proteins in endothelial cells(ECs)can increase intracellular space and BBB leakage.Selective inhibition of mitogen-activated protein kinase,the negative regulatory substrate of mitogen-activated protein kinase phosphatase(MKP)-1,improves tight junction protein function in ECs,and genetic deletion of MKP-1 aggravates ischemic brain injury.However,whether the latter affects BBB integrity,and the cell type-specific mechanism underlying this process,remain unclear.In this study,we established an adult male mouse model of ischemic stroke by occluding the middle cerebral artery for 60 minutes and overexpressed MKP-1 in ECs on the injured side via lentiviral transfection before stroke.We found that overexpression of MKP-1 in ECs reduced infarct volume,reduced the level of inflammatory factors interleukin-1β,interleukin-6,and chemokine C-C motif ligand-2,inhibited vascular injury,and promoted the recovery of sensorimotor and memory/cognitive function.Overexpression of MKP-1 in ECs also inhibited the activation of cerebral ischemia-induced extracellular signal-regulated kinase(ERK)1/2 and the downregulation of occludin expression.Finally,to investigate the mechanism by which MKP-1 exerted these functions in ECs,we established an ischemic stroke model in vitro by depriving the primary endothelial cell of oxygen and glucose,and pharmacologically inhibited the activity of MKP-1 and ERK1/2.Our findings suggest that MKP-1 inhibition aggravates oxygen and glucose deprivation-induced cell death,cell monolayer leakage,and downregulation of occludin expression,and that inhibiting ERK1/2 can reverse these effects.In addition,co-inhibition of MKP-1 and ERK1/2 exhibited similar effects to inhibition of ERK1/2.These findings suggest that overexpression of MKP-1 in ECs can prevent ischemia-induced occludin downregulation and cell death via deactivating ERK1/2,thereby protecting the integrity of BBB,alleviating brain injury,and improving post-stroke prognosis.

人吻素1、脂肪酸结合蛋白质4与糖脂代谢指标的相关性分析及其对妊娠糖尿病的早期诊断价值

目的探讨人吻素1、脂肪酸结合蛋白质4(FABP4)与糖脂代谢指标的相关性及其对妊娠糖尿病(GDM)的早期诊断价值。方法选取2018年1月至2022年1月在河北大学附属医院产科门诊建卡的496例孕妇作为研究对象,根据是否发生GDM分为GDM组和正常组。比较两组孕妇的临床资料。采用Pearson相关分析GDM患者人吻素1、FABP4水平与糖脂代谢指标的相关性。采用多因素Logistic回归分析孕妇发生GDM的危险因素。绘制受试者工作特征(ROC)曲线分析人吻素1、FABP4单独及联合检测对GDM的诊断价值。结果两组孕妇孕前体质量指数(BMI)、孕24周增长体质量、稳态模型胰岛素抵抗指数(HOMA-IR)、稳态模型胰岛β细胞功能指数(HOMA-β)、空腹血糖(FBG)、口服葡萄糖耐量试验(OGTT)1 h血糖(1 hPG)、OGTT 2 h血糖(2 hPG)、糖化血红蛋白(HbA1c)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)、空腹胰岛素(FINS)、人吻素1、FABP4水平比较,差异均有统计学意义(P<0.05)。Pearson相关分析结果显示,GDM孕妇人吻素1水平与孕前BMI、HOMA-IR、FBG、OGTT 1 hPG、OGTT 2 hPG、HbA1c、TG、FINS水平均呈正相关(P<0.05),与HOMA-β呈负相关(P<0.05);GDM孕妇FABP4水平与孕24周增长体质量、HOMA-IR、TG、LDL-C、FINS水平均呈正相关(P<0.05),与高密度脂蛋白胆固醇(HDL-C)水平、HOMA-β均呈负相关(P<0.05)。多因素Logistic回归分析结果显示,孕24周增长体质量、HOMA-IR、HOMA-β、FBG、OGTT 1 hPG、OGTT 2 hPG、HbA1c、TG、FINS、人吻素1、FABP4水平升高是孕妇发生GDM的独立危险因素(P<0.05)。ROC曲线分析结果显示,人吻素1、FABP4联合诊断GDM的曲线下面积(AUC)为0.865,高于人吻素1、FABP4单独诊断GDM的AUC(Z=4.563、5.681,P<0.05)。结论人吻素1、FABP4对GDM的早期诊断具有重要意义,2项指标联合检测能够有效提高GDM的诊断率。

玄参水提取物对高糖暴露条件下INS-1细胞AMPK的激活作用

目的研究玄参水提取物(AESN)对高糖(HG)条件下INS-1细胞腺苷酸活化蛋白激酶(AMPK)活性的影响。方法将INS-1细胞培养于HG培养基中,并给予不同浓度AESN共孵育处理;利用细胞计数试剂-8(CCK-8)法检测AESN干预对细胞增殖/活力和焦亡小体形成的影响,使用Western blotting法观察AESN对细胞内AMPK表达及磷酸化水平的影响;利用时间分辨-荧光共振能量转移(TR-FRET)实验检测AESN对AMPK激酶活性的影响。结果CCK-8检测结果显示同正常培养条件相比,HG暴露显著降低INS-1细胞增殖/活力并增加焦亡小体形成数量,Western blotting检测结果表明HG暴露可导致细胞内AMPK磷酸化水平下降;而AESN共孵育可呈浓度依赖性地增加INS-1细胞增殖/活力、抑制焦亡小体形成并激活AMPK。TR-FRET实验结果表明,AESN可浓度依赖性增加AMPK激酶活性。结论AESN对HG暴露条件下INS-1细胞AMPK具有激活作用。

熊果苷调节PI3K/Akt/GLUT1信号通路对胃癌细胞恶性进展的影响

目的 探讨熊果苷(Arb)调节磷脂酰肌醇-3激酶B/蛋白激酶B/葡萄糖转运蛋白1(PI3K/Akt/GLUT1)信号通路对胃癌细胞恶性进展的影响。方法 使用不同浓度(12.5、25、50、100、200、400 mol/L)Arb处理SNU-601细胞,检测细胞活性,筛选最佳浓度;将细胞分为对照组(Control组)、熊果苷低、中、高浓度组(L-Arb组、M-Arb组、H-Arb组)、熊果苷高浓度+PI3K/Akt激活剂组(H-Arb+740 Y-P组),分别检测细胞凋亡率、细胞周期、葡萄糖摄取、乳酸生成、ATP生成、细胞迁移数和细胞侵袭数;Western blot检测Bax、半胱氨酸天冬氨酸蛋白水解酶3(cleaved-caspase3)、B细胞淋巴瘤-2(Bcl-2)、p-PI3K、PI3K、p-Akt、Akt、GLUT1、HKⅡ蛋白表达。结果 12.5~400 mol/L的Arb可显著抑制SNU-601细胞增殖,选择50、100、200 mol/L的Arb进行后续实验。与Control组比较,L-Arb组、M-Arb组、H-Arb组细胞活性、葡萄糖摄取、乳酸生成、ATP生成、细胞迁移数、细胞侵袭数及Bcl-2、p-PI3K/PI3K、p-Akt/Akt、GLUT1、HKⅡ蛋白表达降低,细胞凋亡率和Bax、cleaved-caspase3蛋白表达增加(P<0.05);与H-Arb组比较,H-Arb+740 Y-P组细胞活性、葡萄糖摄取、乳酸生成、ATP生成、细胞迁移数、细胞侵袭数及Bcl-2、p-PI3K/PI3K、p-Akt/Akt、GLUT1、HKⅡ蛋白表达增加,细胞凋亡率和Bax、cleaved-caspase3蛋白表达降低(P<0.05)。结论 Arb通过抑制PI3K/Akt/GLUT1信号通路抑制胃癌细胞恶性进展。

盘状结构域受体1通过调控NF-κB/NLRP3信号通路介导的细胞焦亡对高糖诱导血管内皮功能障碍的影响

目的 探究盘状结构域受体1 (discoidin domain receptor 1,DDR1)对高糖诱导血管内皮细胞功能障碍的作用机制。方法 体外培养人脐静脉内皮细胞(human umbilical vein endothelial cells,HU VECs),首先分为对照组(Control)、高糖诱导组(high glucose,HG),采用33 mmol·L^(-1) D-glucose处理48 h构建HUVECs功能障碍。碘化丙啶染色(PI)检测细胞焦亡情况;酶联免疫吸附法(ELISA)测定乳酸脱氢酶(lactate dehydrogenase,LDH)、IL-1β、IL-18水平;Western blot检测DDR1、NF-κB/NLRP3信号通路蛋白及细胞焦亡相关蛋白表达;随后,实验分为C ontrol组、HG组、HG+DDR1 NC组、HG+DDR1 siRNA组。转染DDR1 siRNA 24 h后观察HG对HUVECs增殖和迁移能力的影响;ELISA检测内皮型一氧化氮合成酶(endothelial nitric oxide synthase,eNOS)、血管细胞黏附分子-1(vascular cell adhesion molecule-1,VCAM-1)、细胞间黏附分子-1 (intercellular adhesion molecule-1,ICAM-1)及LDH、IL-1β、IL-18水平;PI检测细胞焦亡情况;Western blot检测DDR1、NF-κB/NLRP3信号通路蛋白及细胞焦亡相关蛋白表达。结果 与Control组相比,HG组细胞eNOS含量降低,VCAM-1、ICAM-1含量升高,细胞活力及迁移能力降低,DDR1、p-NF-κB、NLRP3及细胞焦亡相关蛋白表达均明显升高,且LDH、IL-1β、IL-18水平及细胞焦亡率均增加(P<0.05)。与HG组相比,DDR1siRNA能够促进eNOS分泌,降低VCAM-1、ICAM-1、LDH、IL-1β、IL-18水平,增加细胞活力和迁移能力,降低p-NF-κB、NLRP3及细胞焦亡相关蛋白表达,抑制高糖诱导的HU VECs焦亡(P<0.05)。结论基因沉默DDR1能够改善高糖诱导的血管内皮细胞功能障碍,其机制与抑制NF-κB/NLRP3信号通路介导的细胞焦亡相关。

Hexokinase 1 and 2 mediates glucose utilization to regulate the synthesis of kappa casein via ribosome protein subunit 6 kinase 1 in bovine mammary epithelial cells

Glucose plays a vital part in milk protein synthesis through the mTOR signaling pathway in bovine mammary epithelial cells(BMEC).The objectives of this study were to determine how glucose affects hexokinase(HK)activity in BMEC and investigate the regulatory effect of HK in kappa casein(CSN3)synthesis via the mechanistic target of rapamycin complex 1(mTORC1)signaling pathway in BMEC.For this,HK1 and HK2 were knocked out in BMEC using the CRISPR/Cas9 system.The gene and protein expression,glucose uptake,and cell proliferation were measured.We found that glucose uptake,cell proliferation,CSN3 gene expression levels,and expression of HK1 and HK2 increased with increasing glucose concentrations.Notably,glucose uptake was significantly reduced in HK2 knockout(HK2KO)BMEC treated with 17.5 mM glucose.Moreover,under the same glucose treatment conditions,the proliferative ability and abundance of CSN3 were significantly diminished in both HK1 knockout(HK1KO)and HK2KO BMEC compared with that in wild-type BEMC.We further observed that the phosphorylation levels of ribosome protein subunit 6 kinase 1(S6K1)were reduced in HK1KO and HK2KO BMEC following treatment with 17.5 mM glucose.As expected,the levels of glucose-6-phosphate and the m RNA expression levels of glycolysis-related genes were decreased in both HK1KO and HK2KO BMEC following glucose treatment.These results indicated that the knockout of HK1 and HK2 inhibited cell proliferation and CSN3 expression in BMEC under glucose treatment,which may be associated with the inactivation of the S6K1 and inhibition of glycolysis.

血清EPO、HMGB1及GRP78与烧伤患者并发脓毒症的相关性分析

目的 评估血清促红细胞生成素(EPO)、高迁移率族蛋白B1(HMGB1)及葡萄糖调节蛋白78(GRP78)与烧伤后并发脓毒症患者的相关性。方法 选取于徐州医科大学附属宿迁医院2021年1月—2021年12月就诊的烧伤患者作为研究对象,共计110例。根据感染程度分为烧伤后脓毒症组21例、局部感染组47例、未感染组42例。收集患者烧伤面积、糖尿病、高血压患病情况等一般资料。收集外周静脉血,留血清样本采用ELISA法检测EPO、HMGB1及GRP78水平。采用受试者工作曲线(ROC)评估EPO、HMGB1、GRP78对烧伤患者并发脓毒症的预测价值。结果 在烧伤面积方面,局部感染组大于未感染组,而脓毒症组大于局部感染组,差异有统计学意义(P<0.05)。在血清EPO、HMGB1及GRP78水平方面,局部感染组高于未感染组,而脓毒症组高于局部感染组,差异有统计学意义(P<0.05)。EPO预测烧伤患者发生脓毒症的敏感度为76.2%,特异度为80.9%;HMGB1预测烧伤患者发生脓毒症的敏感度为71.4%,特异度为84.3%;GRP78预测烧伤患者发生脓毒症的敏感度为71.4%,特异度为89.9%。结论 烧伤后合并脓毒症患者血清EPO、HMGB1及GRP78水平明显升高,血清EPO、HMGB1及GRP78均对烧伤患者并发脓毒症有较高的预测价值。

米扎格列净通过抑制钠-葡萄糖共转运体1的功能抑制常染色体显性多囊肾细胞增殖和纤维化

目的探究钠-葡萄糖共转运体1(SGLT1)抑制剂米扎格列净(MIZA)在常染色体显性多囊肾病(ADPKD)中的作用。方法用蛋白质印迹法、qPCR、免疫荧光染色测定PKD1^(-/-)小鼠和PKD1^(+/+)小鼠肾脏组织、人肾癌旁组织和人ADPKD组织中SGLT1的表达和分布。用MIZA处理囊肿衬里上皮细胞OX161和肾小管上皮细胞UCL93,37℃孵育24、48和72 h后通过MTT实验和集落形成实验观察细胞增殖情况。以100μmol/L MIZA处理OX161细胞48 h后,通过qPCR测定细胞中α1-Ⅰ型胶原蛋白、α1-Ⅲ型胶原蛋白和纤连蛋白1的mRNA表达量。用犬肾细胞MDCK 3D囊肿形成实验验证MIZA对囊肿形成的作用。通过mRNA-seq数据分析筛选UCL93细胞和OX161细胞、OX161细胞和100μmol/L MIZA处理48 h后的OX161细胞的差异表达基因,利用京都基因与基因组百科全书(KEGG)数据库进行通路富集分析。结果SGLT1在ADPKD患者和PKD1^(-/-)小鼠多囊肾组织中的表达水平较正常肾脏组织升高(P<0.05,P<0.01),免疫荧光染色发现SGLT1主要表达在囊肿衬里上皮细胞。在体外实验中,MIZA呈浓度和时间依赖性地抑制多囊肾细胞的增殖和纤维化,3D形成实验表明MIZA抑制了囊肿的形成。mRNA-seq数据分析和KEGG富集分析结果显示,OX161细胞和100μmol/L MIZA处理48 h的OX161细胞的差异表达基因主要富集在PI3K-Akt、MAPK等信号通路,与OX161细胞和UCL93细胞的差异表达基因富集通路相同。结论SGLT1抑制剂MIZA可能通过PI3K-Akt、MAPK等通路抑制多囊肾细胞的增殖和纤维化,延缓多囊肾的生长,是ADPKD的一个潜在治疗靶点。