Effects of serum containing natural cerebrolysin on glucose-regulated protein 78 and CCAAT enhancer-binding protein homologous protein expression in neuronal PC12 cells following tunicamycin-induced endoplasmic reticulum stress

BACKGROUND： Glucose-regulated protein 78 （GRP78）, a marker of endoplasmic reticulum stress, can prolong cell survival. Alternatively, CCAAT enhancer-binding protein homologous protein （CHOP）, a transcription factor specific for endoplasmic reticulum stress, can cause cell cycle arrest and cell apoptosis. OBJECTIVE： To study the protective effects of serum containing natural cerebrolysin on endoplasmic reticulum stress in tunicamycin-induced neuronal PC12 cells, and analyze the influence on GRP78 and CHOP expressions. DESIGN, TIME AND SETTING： A parallel controlled study was performed at the Institute of Integrated Western and Traditional Chinese Medicine, Shenzhen Hospital, Southern Medical University, between March 2006 and August 2008. MATERIALS： Adult Sprague-Dawley rats were perfused with natural Cerebrolysin aqueous extract （0.185 g/kg/d） to produce serum containing natural Cerebrolysin. Physiological saline was used to produce blank serum. PC12 cell line was provided by Shanghai Institute of Cell Biology, Chinese Academy of Science. Tunicamycin was provided by Sigma （St. Louis, USA）, and natural Cerebrolysin, containing ginseng, rhizoma gastrodiae, and gingko leaf （1：2：2）, by Shengzhen Institute of Integrated Western and Traditional Chinese Medicine. METHODS： PC12 cells were treated with DMEM culture media containing 10% blank serum （normal control group）, tunicamycin （1 μg/mL; model group）, and 5%, 10%, and 15% serum containing natural cerebrolysin and tunicamycin （1 μ g/mL; low-, moderate-, and high-dose serum containing natural cerebrotysin groups）, for 2 hours. MAIN OUTCOME MEASURES： PC12 cells were treated with tunicamycin for 48 hours after which apoptosis was measured using the TUNEL method to calculate apoptotic index. GRP78 expression was detected using immunocytochemistry. After 24 hours of treatment with tunicamycin, GRP78 and CHOP mRNA expressions were measured using RT-PCR. RESULTS： The apoptotic index and CHOP mRNA expression were in the model group and three cerebrolysin groups were significantly increased when compared to the normal control group （P 〈 0.05）. In contrast, GRP78 mRNA and protein expressions were significantly decreased （P 〈 0.05）. CONCLUSION： Serum containing natural cerebrolysin significantly reduced apoptosis in neuronal PC12 cells following tunicamycin-induced endoplasmic reticulum stress. These results may be related to an up-regulation of GRP78 expression and down-regulation of CHOP expression, both of which displayed dose-dependent effects.

Prostaglandin E1 protects hepatocytes against endoplasmic reticulum stress-induced apoptosis via protein kinase A-dependent induction of glucose-regulated protein 78 expression

AIM To investigate the protective effect of prostaglandin E1(PGE1) against endoplasmic reticulum(ER) stressinduced hepatocyte apoptosis, and to explore its underlying mechanisms.METHODS Thapsigargin(TG) was used to induce ER stress in the human hepatic cell line L02 and hepatocarcinomaderived cell line Hep G2. To evaluate the effects of PGE1 on TG-induced apoptosis, PGE1 was used an hour prior to TG treatment. Activation of unfolded protein response signaling pathways were detected by western blotting and quantitative real-time RTPCR. Apoptotic index and cell viability of L02 cells and Hep G2 cells were determined with flow cytometry and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium] assay. RESULTS Pretreatment with 1 μmol/L PGE1 protected against TG-induced apoptosis in both L02 cells and Hep G2 cells. PGE1 enhanced the TG-induced expression of C/EBP homologous protein(CHOP), glucose-regulated protein(GRP) 78 and spliced X box-binding protein 1 at 6 h. However, it attenuated their expressions after 24 h. PGE1 alone induced protein and m RNA expressions of GRP78; PGE1 also induced protein expression of DNA damage-inducible gene 34 and inhibited the expressions of phospho-PKR-like ER kinase, phosphoeukaryotic initiation factor 2α and CHOP. Treatment with protein kinase A(PKA)-inhibitor H89 or KT5720 blocked PGE1-induced up-regulation of GRP78. Further, the cytoprotective effect of PGE1 on hepatocytes was not observed after blockade of GRP78 expression by H89 or small interfering RNA specifically targeted against human GRP78.CONCLUSION Our study demonstrates that PGE1 protects against ER stress-induced hepatocyte apoptosis via PKA pathwaydependent induction of GRP78 expression.

Role of Glucose-regulated Protein 78 in Early Brain Injury after Experimental Subarachnoid Hemorrhage in Rats

Early brain injury（EBI） plays a key role in the pathogenesis of subarachnoid hemorrhage（SAH）. This study investigated the role of glucose-regulated protein 78（GRP78） in EBI after SAH. Male Sprague-Dawley rats（n=108） weighing 260±40 g were divided into control, sham-operated, and operated groups. Blood was injected into the prechiasmatic cistern of rats in the operated group. Neurological scores, ultrastructures of neurons, apoptosis, and GRP78 expression in the hippocampus were examined using Garcia scoring system, transmission electron microscopy, terminal deoxynucleotidyl transferase-mediated d UTP nick-end labelling, and Western blotting at 1, 6, 12, 24, 48, and 72 h after SAH, respectively. The results showed that neurological scores were significantly decreased in the operated group as compared with those in control and sham-operated groups at 12, 24, 48, and 72 h. Metachromatin, chromatin pyknosis at the edge, endoplasmic reticulum swelling, and invagination of nuclear membrane were observed at 24 h in the operated group, indicating the early morphological changes of apoptosis. The number of apoptotic cells was significantly increased in the operated group as compared with that in control and sham-operated groups at 6, 12, 24, 48, and 72 h. The GRP78 protein expression levels in the operated group were significantly elevated at all time points and reached the peak at 12 h. GRP78 expression was positively associated with apoptosis cells and negatively with neurological scores. In conclusion, EBI was demonstrated to occur after SAH and GRP78 was involved in the development of EBI after SAH.

Glucose-regulated protein 78 inhibits scavenger receptor A-mediated internalization of acetylated low density lipoprotein

Class A scavenger receptor(SR-A) plays an important role in foam cell formation.However, the mechanism underlying the internalization of the receptor-ligand complexes remains unclear.The aim of the present study was to investigate the molecular mechanism to regulate SR-A-mediated intracellular lipid accumulation in macrophages A pull-clown assay was performed and glucoseregulated protein 78(GRP78) was identified to bind with the cytoplasmic domain of SR-A(CSR-A).Immunoprecipitation and artificially expressed protein binding assay demonstrated the direct specific binding of GRP78 with SR-A in cells.Indirect immunofluorescence assay and western blot analysis showed their co-localization in membrane and cytoplasm.Over-expression of GRP78 specifically inhibited SR-A-mediated uptake of fluorescent acetylated low-density lipoprotein, a specific ligand for SR-A, without altering cellular SR-A expression and binding ability, and significantly inhibited cholesterol ester accumulation in cells, which can be partly attributed to the suppression of c-Jun-NH2-terminal kinase signaling pathway.These results suggest that GRP78 may act as an inhibitor of SR-A-mediated internalization of modified low-density lipoprotein into macrophages(C) 2009 Elsevier Inc.All rights reserved.

Physiological roles of mitogen-activated-protein-kinase-activated p38-regulated/activated protein kinase

Mitogen-activated protein kinases(MAPKs)are a family of proteins that constitute signaling pathways involved in processes that control gene expression,cell division, cell survival,apoptosis,metabolism,differentiation and motility.The MAPK pathways can be divided into conventional and atypical MAPK pathways.The first group converts a signal into a cellular response through a relay of three consecutive phosphorylation events exerted by MAPK kinase kinases,MAPK kinase,and MAPK.Atypical MAPK pathways are not organized into this three-tiered cascade.MAPK that belongs to both conventional and atypical MAPK pathways can phosphorylate both non-protein kinase substrates and other protein kinases.The latter are referred to as MAPK-activated protein kinases.This review focuses on one such MAPK-activated protein kinase,MAPK-activated protein kinase 5(MK5)or p38-regulated/activated protein kinase(PRAK).This protein is highly conserved throughout the animal kingdom and seems to be the target of both conventional and atypical MAPK pathways.Recent findings on the regulation of the activity and subcellular localization,bona fide interaction partners and physiological roles of MK5/PRAK are discussed.

Regulation of glucose and protein syntheses by Micrococcus luteus during the fermentation of a Nigerian rice, <i>Oryza sativa</i>variety “Igbimo”

Micrococcus luteus synthesises glucose and protein during the fermentation of a Nigerian rice. To regulate the formation of these substances, mutation was carried out with an alkylating agent: ethylmethyl sulphonate (EMS). Screening the mutants generated for the levels of the traits expressed, four major groups were obtained. These are poor, moderate, good and super producers of either glucose or protein. They produced the properties at 0 - 1.00, 1.01 to 1.99 (moderate) and 2.0 to 2.99 (good) and 3.0 and above (super) mg.mL–1 of each substance. The classes were made up of 37, 40, 20 and 3 mutants for glucose production and 13, 35, 40 and 12 mutants for protein synthesis. The wild strain bacterium made 0.86 mg.mL–1 glucose and 1.2 mg.mL–1 protein describing the M. luteus as poor glucose maker and moderate protein producer. It was also noticed that the mutation caused some variants (25%) to form more glucose than protein;the remaining 75% of the population are made up of two sets viz: mutants having better ability to synthesise protein at higher concentrations than glucose and those that formed about the same amounts of the substances. It thus follows that the glucose and protein productions in M. luteus are genetically based and can be regulated by genetic manipulation.

MiR-183-5p promotes the progression of non-small cell lung cancer through targeted regulation of FOXO1

Objective To investigate miR-183-5p targeting to forkhead box protein O1(FOXO1)and its corresponding effect on the proliferation,migration,invasion,and epithelial-mesenchymal transition(EMT)of non-small cell lung cancer(NSCLC)cells.Methods NSCLC tissues and adjacent normal tissues from 60 patients with NSCLC adenocarcinoma were obtained via pathological biopsy or intraoperative resection.Several cell lines were cultured in vitro,including the human normal lung epithelial cell line BEAS-2B and human NSCLC cell lines A549,SPCA-1,PC-9,and 95-D.miR-183-5p and FOXO1 mRNA expression in tissues and cells were detected by qRT-PCR;the corresponding correlations in NSCLC tissues were analyzed using the Pearson test,and the relationship between miR-183-5p expression and clinicopathological parameters was analyzed.The miR-183-5p-mediated regulation of FOXO1 was verified by bioinformatics prediction alongside double luciferase,RNA-binding protein immunoprecipitation(RIP)assay,and pull-down experiments.A549 cells were divided into control,anti-miR-NC,anti-miR-183-5p,miR-NC,miR-183-5p,miR-183-5p+pcDNA3.1,and miR-183-5p+pcDNA3.1-FOXO1 groups.Cell proliferation,invasion,migration,apoptosis,and cell cycle distribution were detected using an MTT assay,clone formation assay,Transwell assay,scratch test,and flow cytometry,respectively.The expression of EMT-related proteins in the cells was analyzed by western blotting.The effect of miR-185-3p silencing on the development of transplanted tumors was detected by analyzing tumor formation in nude mice.Results miR-183-5p expression was significantly higher in NSCLC tissues and cells than in adjacent normal tissues,whereas FOXO1 mRNA expression was significantly down-regulated.There was a significant negative correlation between miR-183-5p and FOXO1 mRNA in NSCLC tissues(P<0.05).Additionally,the expression of miR-183-5p was significantly correlated with tumor size,tumor differentiation,and tumor-node-metastasis stage in patients with NSCLC(P<0.05).miR-183-5p targeted and inhibited FOXO1 expression.Compared to the anti-miR-NC group,the cell proliferation,scratch healing rate,N-cadherin and vimentin protein expression,and the proportion of S phase cells were significantly lower in the anti-miR-183-5p group,whereas the protein expression of E-cadherin andα-catenin and the proportion of G0/G1 phase cells were significantly higher;additionally,the frequency of colony formation and invasion were significantly lower in the anti-miR-183-5p group(P<0.05).Compared to the miR-NC group,the cell proliferation,scratch healing rate,N-cadherin and vimentin protein expression,and the proportion of S phase cells in the miR-183-5p group were significantly higher,whereas the E-cadherin andα-catenin protein expression and the proportion of G0/G1 phase cells were significantly lower;furthermore,the frequency of colony formation and invasion were significantly higher in the miR-183-5p group(P<0.05).Compared with the miR-183-5p+pcDNA3.1 group,the OD value,scratch healing rate,N-cadherin and vimentin protein expression,and the proportion of S phase cells were significantly lower in the miR-183-5p+pcDNA3.1-FOXO1 group,whereas E-cadherin andα-catenin protein expression and the proportion of G0/G1 phase cells were significantly higher;additionally,the frequency of colony formation and invasion was significantly lower in the miR-183-5p+pcDNA3.1-FOXO1 group(P<0.05).Overall,silencing miR-185-3p inhibited the growth of transplanted tumors and promoted FOXO1 expression.Conclusion Overexpression of miR-183-5p can inhibit apoptosis and promote the proliferation,migration,invasion,and EMT,of NSCLC cells by down-regulating FOXO1 expression.

过表达葡萄糖调节蛋白78人乳腺癌细胞系HS578T、MDA-MB-231的构建

目的构建过表达内质网应激标志蛋白葡萄糖调节蛋白78(Glucose-regulated protein 78,GRP78)的人乳腺癌细胞系HS578T、MDA-MB-231,为探讨GRP78在乳腺癌发生发展中的作用机制提供基础。方法采用分子克隆技术构建表达GRP78的慢病毒载体pCDH-CMV-GRP78-HA-EF1-Puro质粒,采用PCR法扩增质粒,后使用EcoRⅠ、NotⅠ限制性内切酶切割质粒,对酶切产物进行测序鉴定,成功构建pCDH-CMV-GRP78-HA-EF1-Puro质粒。取对数生长期293FT细胞系(克隆分离株),在培养皿中加入pCDH-CMV-GRP78-HA-EF1-Puro混合液,培养48 h收集含GRP78慢病毒颗粒的上清液,保存备用。另取对数生长期人乳腺癌细胞系HS578T及MDA-MB-231,置于含GRP78慢病毒颗粒上清液的培养基中培养,培养48 h时在培养基中加入1∶500稀释嘌呤霉素,筛选GRP78过表达的HS578T、MDA-MB-231细胞系。加入嘌呤霉素培养48 h采用Western Blotting法检测HS578T、MDA-MB-231细胞GRP78。采用激光共聚焦显微镜对HS578T、MDA-MB-231细胞中GRP78蛋白进行定位。结果构建表达GRP78的慢病毒载体pCDH-CMV-GRP78-HA-EF1-Puro质粒。GRP78过表达的HS578T、MDA-MB-231细胞系在78 kD左右可见明显GPR78蛋白电泳条带。HS578T细胞系中存在GRP78蛋白表达,主要分布在细胞质中。结论成功构建GRP78过表达的MDA-MB-231、HS578T细胞系,GRP78主要表达于细胞质中。

GRP78-c-Src信号通路介导周期性牵张力作用下牙周膜成纤维细胞成骨分化的机制研究

目的探讨在周期性牵张力作用下葡萄糖调节蛋白78(GRP78)对牙周膜成纤维细胞成骨分化的影响,并阐述其机制。方法应用FlexCell 5000细胞应力装置模拟牙齿正畸受力环境;应用流式细胞术细胞分选技术获得牙周膜成纤维细胞GRP78高表达细胞和GRP78低表达细胞;采用基因转染技术敲减GRP78、c-Src的表达以及过表达c-Src;蛋白质印迹实验检测成骨转录因子Runt相关基因2(RUNX2)、锌指结构转录因子(Osterix)以及成骨标志蛋白骨钙蛋白(OCN)、骨桥蛋白(OPN)的表达;免疫共沉淀实验检测GRP78与c-Src激酶的相互作用;茜素红染色实验检测细胞矿化结节的形成。结果GRP78在牙周膜成纤维细胞呈异质性表达,流式分选实验获得GRP78高表达和GRP78低表达细胞。周期性牵张力处理后,与GRP78低表达细胞相比,GRP78高表达细胞成骨分化能力及c-Src激酶磷酸化水平均升高,差异具有统计学意义(P<0.05);GRP78与c-Src激酶存在相互作用;与对照组相比,过表达c-Src组细胞成骨分化能力升高(P<0.05),sic-Src组细胞成骨分化能力降低(P<0.05)。结论GRP78通过与c-Src激酶相互作用并上调其表达,进而促进周期性牵张力诱导的牙周膜成纤维细胞成骨分化。

溃疡性结肠炎患儿血清sB7-H3、GRP78水平及与病情、预后的关系

目的探究溃疡性结肠炎(UC)患儿血清可溶性B7同源体3(sB7-H3)、葡萄糖调节蛋白78(GRP78)水平及与病情、预后的关系。方法选取2019年8月至2020年8月在本院诊治的UC患儿78例作为研究组,选取同时期在本院进行健康体检的儿童75例作为对照组。根据研究组病情严重情况分为轻度组29例,中度组32例,重度组17例。对研究组进行3年随访,根据预后情况分为预后不良组22例,预后良好组56例。采用酶联免疫吸附法测定血清sB7-H3、GRP78及炎症因子干扰素-r(IFN-r)、白细胞介素-1β(IL-1β)、白细胞介素-33(IL-33)的水平;根据血压测量仪检测舒张压和收缩压水平。Pearson法分析血清sB7-H3和GRP78分别与炎症因子的相关性;Logistic回归分析影响UC患儿预后发生不良的因素;受试者工作特征(ROC)曲线分析血清sB7-H3和GRP78水平预测UC患儿预后发生不良的临床价值。结果研究组血清sB7-H3、GRP78、IFN-r、IL-1β、IL-33水平显著高于对照组(P<0.05)。随着UC患儿病情越严重,血清sB7-H3、GRP78、IFN-r、IL-1β、IL-33水平越高(P<0.05)。血清sB7-H3和GRP78分别与IFN-r、IL-1β、IL-33呈正相关(P<0.05)。不良组血清sB7-H3、GRP78、IFN-r、IL-1β、IL-33水平显著高于良好组(P<0.05)。血清sB7-H3、GRP78、IL-33是影响UC患儿预后发生不良的危险因素(P<0.05)。血清sB7-H3、GRP78及二者联合检测UC患儿预后发生不良的曲线下面积(AUC)为0.874、0.884、0.969,二者联合检测明显高于单独检测(Z二者联合-sB7-H3=1.878,Z二者联合-GRP78=1.931,P<0.05)。结论UC患儿血清sB7-H3、GRP78水平升高,其随病情越严重水平越高,二者联合可较好预测UC患儿预后发生不良的情况。

LncRNA TUG1通过调节miR-181b-5p/PDCD4轴对高糖诱导的心肌细胞凋亡的影响

目的 探讨长链非编码RNA(LncRNA)牛磺酸上调基因1(TUG1)通过调节miR-181b-5p/程序性细胞死亡蛋白4(PDCD4)轴对高糖诱导的心肌细胞凋亡的影响。方法 采用高糖(25 mmol/L葡萄糖)在体外构建糖尿病心肌病(DCM)细胞模型。AC16细胞分为NG组、HG组、HG+sh-NC组、HG+sh-TUG1组、HG+miR-NC组、HG+miR-181b-5p组、HG+sh-TUG1+anti-miR-NC组、HG+sh-TUG1+anti-miR-181b-5p组、HG+miR-181b-5p+pcDNA组、HG+miR-181b-5p+pc-PDCD4组。细胞计数试剂盒-8(CCK-8)法检测细胞活力;乳酸脱氢酶(LDH)测定试剂盒检测LDH释放总量;采用实时定量聚合酶链反应(q RT-PCR)检测TUG1、miR-181b-5p和PDCD4 mRNA表达;流式细胞术检测细胞凋亡;Western blot检测B细胞淋巴瘤2-相关X(Bax)、活化的胱天蛋白酶3(cleaved caspase 3)和PDCD4蛋白表达;caspase-Glo3检测试剂盒评估caspase 3活性;双萤光素酶报告基因实验验证TUG1或PDCD4与miR-181b-5p的靶向关系。结果 与NG组比较,HG组细胞活性降低,LDH释放总量、凋亡率、Bax、cleaved caspase 3表达及caspase 3活性升高(P<0.05),敲低TUG1或上调miR-181b-5p表达可拮抗上述变化(P<0.05)。抑制miR-181b-5p可减轻TUG1沉默对高糖处理下心肌细胞活力和凋亡的影响(P<0.05)。PDCD4过表达可减弱miR-181b-5p上调对高糖处理的心肌细胞活力的促进作用和对凋亡的抑制作用。TUG1可通过吸附miR-181b-5p上调PDCD4表达(P<0.05)。结论TUG1通过下调miR-181b-5p、上调PDCD4表达促进高糖诱导的心肌细胞凋亡。

miRNA-126调控自噬减轻高糖诱导血管内皮细胞损伤的作用机制

目的探讨miRNA-126对高糖诱导的内皮细胞自噬与凋亡的影响及分子机制。方法采用高糖(HG)诱导HUVECs模型,随机分为4组,即正常葡萄糖(5.5 mmo/L)对照组,HG模型组,miRNA-126-5p mimics组(转染miRNA-126-5p mmics组),miRNA-126-5p inhibitor组(转染miRNA-126 inhibitor组)。HG模型组即终浓度为33.3 mmol/L的葡萄糖诱导24 h,miRNA-126-5p mimics组是miRNA-126-5p mimics转染至HUVECs细胞,48 h后在培养基中加入33.3 mmol/L(终浓度)葡萄糖,培养24 h。miRNA-126-5p inhibitor组是miRNA-126-5p inhibitor转染至HUVECs细胞,48 h后在培养基中加入33.3 mmol/L(终浓度)的葡萄糖,培养24 h。再利用miRNA-126-5p mimics组模型,分别予自噬抑制剂3-甲基腺嘌呤(3-methyl adenine,3-MA)5 mmol/L、ERK通路抑制剂(U-0126)10 mmol/L干预24 h。RT-qPCR检测细胞中miRNA-126-5p的表达水平,CCK-8检测细胞活性,划痕实验检测细胞迁移能力,Westernblot检测细胞培养液中血管内皮生长因子(VEGF)和肝细胞生长因子(HGF),p-ERK、ERK蛋白,p-AKT、AKT蛋白,Bax、Bcl-2蛋白,cleaved-caspase-3蛋白,Beclin-1和Lc3蛋白含量。结果与正常组比较,HG模型细胞中miRNA-126水平,p-ERK/ERK蛋白水平低于正常对照组(P<0.05);与HG模型组相比较,miRNA-126-5p mimics组细胞中miRNA-126、p-ERK/ERK蛋白表达明显升高(P<0.01),细胞中Bax/Bcl-2蛋白,cleaved caspase-3蛋白表达下降(P<0.05),Beclin-1和Lc3蛋白的表达水平升高(P<0.01),细胞活性增强(P<0.01),细胞迁移率提高(P<0.01),VEGF水平升高(P<0.05);与miRNA-126-5p inhibitor组比较,miRNA-126-5p mimics组细胞中miRNA-126、pERK/ERK蛋白表达升高(P<0.01),细胞中Bax/Bcl-2蛋白,cleaved caspase-3蛋白表达下降(P<0.05),Beclin-1和Lc3蛋白表达水平升高(P<0.01),细胞活性增强(P<0.05),细胞迁移率提高(P<0.05),VEGF水平升高(P<0.01);在miRNA-126-5p mimics组中加入3-MA后凋亡蛋白表达明显下降(P<0.01),加入ERK抑制剂后,自噬蛋白表达明显下降(P<0.01)。结论miRNA-126可通过激活ERK信号通路促进细胞的自噬并抑制细胞凋亡,减轻HG诱导的内皮细胞损伤。

葡萄糖调节蛋白94和转化生长因子-β1与房颤患者预后的相关性

目的探讨葡萄糖调节蛋白94(glucose-regulated protein 94,GRP94)、转化生长因子-β1(transforming growth factor-β1,TGF-β1)水平对房颤患者消融术后远期复发事件的预测价值。方法选择因房颤行射频消融术的住院患者共202例,统一归于房颤组,再依据房颤发生及持续的时间进一步分为阵发性房颤组(n=132)和持续性房颤组(n=70);另选择窦性心律且既往无房颤心律的健康体检者作为对照组(n=55)。通过酶联免疫吸附试验测定患者的血清GRP94及TGF-β1水平,并收集基线资料。出院后以门诊或电话形式对房颤患者进行随访,利用受试者工作特征曲线和Kaplan-Meier生存分析评估血清GRP94、TGF-β1水平对房颤患者消融术后远期复发事件的预测价值。结果在对照组、阵发性房颤组、持续性房颤组中,血清GRP94及TGF-β1水平依次升高。二元Logistic回归分析提示,血清TGF-β1、GRP94和体重指数是房颤发生的独立危险因素。Spearman相关性分析显示,在房颤患者血清中GRP94和TGF-β1水平呈正相关。复发组的TGF-β1高于非复发组,GRP94低于非复发组。二元Logistic回归分析表明,TGF-β1是房颤术后复发的独立危险因素,GRP94是房颤复发的独立保护因素。Kaplan-Meier曲线分析显示,GRP94高水平、TGF-β1低水平的房颤患者消融术后复发的风险低,且未复发时间更长。结论GRP94及TGF-β1水平与房颤密切相关,可能为房颤患者消融术后复发的早期识别、诊断及预防提供依据。

MOTS-c联合GRP78预测急性心肌梗死患者PCI术后MACE发生的价值

目的探讨MOTS-c和葡萄糖调节蛋白78(GRP78)表达水平对心肌梗死患者经皮冠状动脉介入治疗(PCI)术后主要不良心血管事件(MACE)预测价值。方法选择2019年10月至2022年10月到驻马店市中心医院进行PCI手术治疗的102例急性心肌梗死患者,所有患者均进行为期6个月随访,依据患者MACE发生情况将患者分为MACE组和非MACE组,其中MACE组患者有39例,非MACE组患者63例。应用查询病历和调查问卷等方式对两组患者的一般资料(年龄、性别、心率、体重指数、收缩压、舒张压、糖尿病史和心力衰竭病史)进行统计。应用酶联免疫吸附法对患者血清GRP78和MOTS-c水平进行检测。结果MACE组和非MACE组患者的糖尿病史、体重指数、年龄和性别差异无统计学意义(P>0.05),但是MACE组患者的收缩压和舒张压比非MACE组低(P<0.05),且MACE组患者的心力衰竭病史率和心率比非MACE组高(P<0.05)。MACE组患者的GRP78水平均比非MACE组患者高(P<0.05),而MACE组患者的MOTS-c水平低于非MACE组患者(P<0.05)。GRP78和MOTS-c预测的曲线下面积(AUC)为0.889和0.721,截断值分别为1.313、183.27μg·L^(-1),其灵敏度分别为76.48%和63.29%,特异度分别为98.21%和91.38%,两者联合诊断评估MACE的AUC为0.926,灵敏度为80.93%,特异度为97.32%。经过logistic多因素分析,GRP78>1.313μg·L^(-1)、MOTS-c≤183.27μg·L^(-1)和存在心力衰竭病史是急性心肌梗死患者PCI术后MACE发生的独立危险因素(P<0.05)。结论急性心肌梗死患者PCI术后MACE发生患者的GRP78水平升高,MOTS-c水平降低,GRP78联合MOTS-c对急性心肌梗死患者PCI术后MACE发生有预测价值。

血清sCD14-st、CD163和GRP78对急性胰腺炎预后不良的诊断价值

目的探讨血清可溶性白细胞分化抗原14亚型(sCD14-st)、CD163和葡萄糖调节蛋白78(GRP78)对急性胰腺炎预后不良的诊断价值。方法选择2020年8月至2022年6月上海市嘉定区江桥医院和上海市普陀区利群医院收治的急性胰腺炎患者123例作为急性胰腺炎组,另选择同期在上海市嘉定区江桥医院和上海市普陀区利群医院进行健康体检者45例作为健康对照组。观察两组研究对象血清sCD14-st、CD163、GRP78水平变化情况,采用单因素和多因素分析预后不良的影响因素,以及血清sCD14-st、CD163和GRP78水平检测对急性胰腺炎严重程度和预后不良的诊断效能。结果急性胰腺炎组患者血清sCD14-st、CD163、GRP78水平均高于健康对照组,且随着急性胰腺炎严重程度升高而升高,差异均有统计学意义(P<0.05)。急性胰腺炎组中预后不良患者血清D-二聚体、C反应蛋白、sCD14-st、CD163、GRP78水平,以及急性生理和慢性健康状况评分Ⅱ(APACHEⅡ)均高于预后良好患者,差异均有统计学意义(P<0.05);而预后不良患者年龄、性别、体质量指数、淀粉酶、空腹血糖、甘油三酯水平与预后良好患者比较,差异均无统计学意义(P>0.05)。APACHEⅡ≥25.10分、sCD14-st≥3.41 ng/mL、CD163≥718.56 ng/mL、GRP78≥424.98 ng/mL是急性胰腺炎预后不良的危险因素(P<0.05)。血清sCD14-st、CD163、GRP78水平检测对急性胰腺炎预后不良具有较高的诊断效能,3项指标联合检测的灵敏度、特异度、受试者工作特征曲线下面积(AUC)均高于单项指标,差异均有统计学意义(P<0.05);而3项指标单项检测的AUC比较,差异无统计学意义(P>0.05)。结论血清sCD14-st、CD163、GRP78水平是诊断急性胰腺炎严重程度的指标,是发生预后不良的独立影响因素,3项指标联合检测有助于提高对急性胰腺炎预后不良的诊断效能。

低浓度氟化钠对人牙髓细胞的成骨/成牙本质分化的影响

目的探讨低浓度氟化钠对人牙髓细胞(human dental pulp cells,hDPCs)成骨/成牙本质分化的影响。方法本研究已通过单位伦理委员会审查批准。原代培养hDPCs,采用MTT法检测不同浓度氟化钠对hDPCs增殖的影响;选取合适浓度的氟化钠加入成骨/成牙本质分化诱导培养液中,对hDPCs进行体外诱导,通过茜素红染色检测hDPCs成骨/成牙本质分化能力的变化,RT⁃qPCR检测分化相关基因的mRNA表达;同时通过RT⁃qPCR和Western blot检测hDPCs成骨/成牙本质分化过程中内质网应激相关基因的表达。结果低浓度氟化钠(0.1 mmol/L)在体外可刺激hDPCs增殖,高浓度氟化钠(5~10 mmol/L)可抑制hDPCs增殖(P<0.05)。选取0.1 mmol/L氟化钠体外混合成骨/成牙本质分化诱导培养后hDPCs的茜素红染色增加,成骨/成牙本质分化相关基因牙本质涎磷蛋白(dentin sialophosphoprotein,DSPP)、骨涎蛋白(bone sialoprotein,BSP)和骨钙蛋白(osteocalcin,OCN)mRNA表达水平升高(P<0.05)。同时在此过程中RT⁃qPCR检测出mRNA水平hDPCs内质网应激相关基因:剪切x盒结合蛋白1(splicing x⁃box binding protein⁃1,sXBP1)、葡萄糖调节蛋白78(glucose⁃regulated protein 78,GRP78)以及活化转录因子4(activating transcription factor 4,ATF4)表达升高(P<0.05);Western blot检测出氟化钠混合成骨/成牙本质分化培养后细胞磷酸化真核起始因子⁃2α(phosphorylated eukary⁃otic initiation factor⁃2α,p⁃eIF2α)、磷酸化蛋白激酶样内质网激酶(phosphorylated the RNA⁃activated protein kinase⁃like ER⁃resident kinase,p⁃PERK)和ATF4蛋白表达增加(P<0.05)。结论低剂量氟化钠促进人牙髓细胞的成骨/成牙本质分化并伴有内质网应激水平的升高。

绝经后骨质疏松症患者外周血中GRP78和ATF4的表达水平及临床意义

目的探究绝经后骨质疏松症(postmenopausal osteoporosis,PMO)患者外周血中葡萄糖调节蛋白78(glucoseregulated protein 78,GRP78)和激活转录因子4(activating transcription factor 4,ATF4)的水平及临床意义。方法选取2018年12月—2021年5月石家庄市第三医院创伤骨科收治的96例PMO患者为PMO组,另选取同期96名体检健康的绝经女性为正常组。比较PM O组和正常组的一般资料;采用酶联免疫吸附法测定血清GR P78、ATF4水平;利用电化学发光免疫分析仪测定血清β-胶原特殊序列(β-Crosslaps)、Ⅰ型前胶原氨基端前肽(procollagenⅠtype N-terminal propeptide,PINP)水平;利用双能X线骨密度仪检测腰椎骨密度(bone mineral density,BMD);采用Pearson法分析PMO患者血清GRP78、ATF4水平与β-Crosslaps、PIN P、BM D的相关性及PMO患者血清GR P78表达水平与ATF4的相关性;用Logistic回归分析PMO发生的影响因素。结果PMO组患者腰臀比、BMD低于正常组(t=9.76、15.47,P均<0.001);血清GRP78、ATF4、β-Crosslaps、PINP水平高于正常组(t=18.32、16.81、8.15、14.84,P均<0.001);PMO患者血清GRP78、ATF4水平与β-Crosslaps、PINP均呈正相关(r=0.56、0.48、0.48、0.52,P<0.001、<0.001、<0.001、=0.006),与BMD呈负相关(r=-0.54、-0.44,P均<0.001);PMO患者血清GRP78水平与ATF4呈正相关(r=0.595,P<0.001);PINP、GRP78、β-Crosslaps、ATF4是影响PMO发生的危险因素(OR=2.518、2.672、2.271、2.336,P均<0.001),BMD是影响PMO发生的保护因素(OR=0.583,P<0.001)。结论PMO患者血清GRP78、ATF4水平较高,均与β-Crosslaps、PINP、BMD相关,GRP78、ATF4可能是诊治PMO的潜在靶标,测定血清GRP78、ATF4水平有助于临床防治PMO。

血清ADAM15、GRP78联合CT肺动脉造影对急性肺栓塞的诊断价值

目的探究血清整合素金属蛋白酶15(ADAM15)、葡萄糖调节蛋白78(GRP78)联合CT肺动脉造影(CTPA)对急性肺栓塞(APE)的诊断价值。方法选取本院2021年6月至2023年6月收治的86例疑似APE患者,患者均行CTPA检查;根据病情严重程度将APE患者分为中高危组和低危组;酶联免疫吸附法检测ADAM15、GRP78水平;Pearson相关性分析血清ADAM15、GRP78与CTPA指标的相关性;中高危APE的影响因素采用多因素Logistic回归分析;绘制ROC曲线分析血清ADAM15、GRP78对中高危APE的诊断价值。结果86例患者经过CTPA检测出栓子702个,86例患者在不同肺动脉部位表现出充盈缺损等,病变部位主要位于双肺29例,左肺30例,右肺27例。4种栓塞类型42例中心型,98例偏心型,26例附壁血栓型,30例完全堵塞型。中高危组RVD/LVD、RV-LD/LV-LD、Qanadli栓塞指数显著高于低危组(P<0.05)。中高危组血清ADAM15、GRP78水平显著高于低危组(P<0.05)。根据Pearson相关性分析得知,血清ADAM15与GRP78呈正相关(P<0.05),二者均与RVD/LVD、RVLD/LV-LD、Qanadli栓塞指数呈正相关(P<0.05)。多因素Logistic回归分析得知ADAM15、GRP78、RVD/LVD、RV-LD/LV-LD、Qanadli栓塞指数为影响中高危APE患者的危险因素(P<0.05)。根据ROC曲线得知,血清ADAM15、GRP78、RVD/LVD、RVLD/LV-LD和Qanadli栓塞指数五者联合诊断中高危APE的AUC为0.990,五者联合优于各自单独诊断(Z_(联合vs ADAM15)=2.691、Z_(联合vs GRP78)=2.578、Z_(联合vs RVD/LVD)=2.710、Z_(联合vs RV-LD/LV-LD)=2.714、Z联合vs Qanadli栓塞指数=2.698,P均<0.05)。结论血清ADAM15、GRP78在APE患者中显著升高,二者联合CTPA可提高对APE的诊断价值。

牛磺酸对肾NRK-52E细胞缺氧/复氧损伤的保护及初步机制

目的观察牛磺酸(Tau)对NRK-52E细胞缺氧/复氧(H/R)损伤的保护作用及初步机制。方法用NRK-52E细胞缺氧8 h复氧12 h培养建立了H/R模型,流式细胞仪检测凋亡率和胞内游离钙离子浓度,RT-PCR和Western blot检测GRP78、Caspase-12、Caspase-3 mRNA和蛋白表达。结果与对照组比较,H/R组细胞凋亡率上升,GRP78、Caspase-12及Caspase-3 mRNA和蛋白表达均明显增高(P<0.01),胞质游离钙离子浓度也升高(P<0.01);与H/R组比较,各浓度牛磺酸处理组的细胞Caspase-3活性和凋亡率明显下降,胞质游离钙和GRP78、Caspase-12及Caspase-3 mRNA和蛋白表达均有明显降低(P<0.01)。结论牛磺酸对NRK-52E细胞H/R损伤有较好的抑制作用,且呈一定的剂量依赖性,其机制可能是调节胞内钙稳态、抑制GRP78、Caspase-12及Caspase-3表达,减少了细胞凋亡。

牛磺酸对缺血/再灌注大鼠肾脏GRP78和Caspase-12表达的影响

观察牛磺酸(Tau)对肾缺血/再灌注(I/R)大鼠肾脏GRP78、Caspase-12表达的影响及其意义。将Wistar大鼠30只随机分为假手术组(对照组)、I/R组和I/R+TMP组。采用夹闭双侧肾蒂45min再灌注24h制备肾I/R模型。I/R+TMP组在手术前1h按200mg.kg-1腹腔注射Tau,余操作同I/R组。检测血清尿素氮(BUN)和肌酐(Cr);光镜观察肾小管组织结构变化;RT-PCR和免疫组化检测GRP78、Caspase-12mRNA和蛋白表达。与假手术组比较,I/R组大鼠血清BUN,Cr水平显著升高,肾组织损伤严重,GRP78和Caspase-12mRNA和蛋白表达呈显著性增加(P<0.01);与I/R组比较,I/R+Tau组血清BUN、Cr水平及GRP78、Caspase-12表达均有显著性下降(P<0.05),肾脏损伤明显减轻。牛磺酸对肾脏缺血/再灌注大鼠GRP78、Caspase-12过度表达升高有很好的抑制作用,这可能是它减轻肾脏缺血/再灌注损伤的重要机制之一。