Endoplasmic reticulum stress and autophagy in cerebral ischemia/reperfusion injury:PERK as a potential target for intervention

Several studies have shown that activation of unfolded protein response and endoplasmic reticulum(ER)stress plays a crucial role in severe cerebral ischemia/reperfusion injury.Autophagy occurs within hours after cerebral ischemia,but the relationship between ER stress and autophagy remains unclear.In this study,we established experimental models using oxygen-glucose deprivation/reoxygenation in PC12 cells and primary neurons to simulate cerebral ischemia/reperfusion injury.We found that prolongation of oxygen-glucose deprivation activated the ER stress pathway protein kinase-like endoplasmic reticulum kinase(PERK)/eukaryotic translation initiation factor 2 subunit alpha(e IF2α)-activating transcription factor 4(ATF4)-C/EBP homologous protein(CHOP),increased neuronal apoptosis,and induced autophagy.Furthermore,inhibition of ER stress using inhibitors or by si RNA knockdown of the PERK gene significantly attenuated excessive autophagy and neuronal apoptosis,indicating an interaction between autophagy and ER stress and suggesting PERK as an essential target for regulating autophagy.Blocking autophagy with chloroquine exacerbated ER stress-induced apoptosis,indicating that normal levels of autophagy play a protective role in neuronal injury following cerebral ischemia/reperfusion injury.Findings from this study indicate that cerebral ischemia/reperfusion injury can trigger neuronal ER stress and promote autophagy,and suggest that PERK is a possible target for inhibiting excessive autophagy in cerebral ischemia/reperfusion injury.

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.

Cell division cyclin 25C knockdown inhibits hepatocellular carcinoma development by inducing endoplasmic reticulum stress

BACKGROUND Cell division cyclin 25C(CDC25C)is a protein that plays a critical role in the cell cycle,specifically in the transition from the G2 phase to the M phase.Recent research has shown that CDC25C could be a potential therapeutic target for cancers,particularly for hepatocellular carcinoma(HCC).However,the specific regulatory mechanisms underlying the role of CDC25C in HCC tumorigenesis and development remain incompletely understood.AIM To explore the impact of CDC25C on cell proliferation and apoptosis,as well as its regulatory mechanisms in HCC development.METHODS Hepa1-6 and B16 cells were transduced with a lentiviral vector containing shRNA interference sequences(LV-CDC25C shRNA)to knock down CDC25C.Subsequently,a xenograft mouse model was established by subcutaneously injecting transduced Hepa1-6 cells into C57BL/6 mice to assess the effects of CDC25C knockdown on HCC development in vivo.Cell proliferation and migration were evaluated using a Cell Counting Kit-8 cell proliferation assays and wound healing assays,respectively.The expression of endoplasmic reticulum(ER)stress-related molecules(glucose-regulated protein 78,X-box binding protein-1,and C/EBP homologous protein)was measured in both cells and subcutaneous xenografts using quantitative real-time PCR(qRT-PCR)and western blotting.Additionally,apoptosis was investigated using flow cytometry,qRT-PCR,and western blotting.RESULTS CDC25C was stably suppressed in Hepa1-6 and B16 cells through LV-CDC25C shRNA transduction.A xenograft model with CDC25C knockdown was successfully established and that downregulation of CDC25C expression significantly inhibited HCC growth in mice.CDC25C knockdown not only inhibited cell proliferation and migration but also significantly increased the ER stress response,ultimately promoting ER stress-induced apoptosis in HCC cells.CONCLUSION The regulatory mechanism of CDC25C in HCC development may involve the activation of ER stress and the ER stress-induced apoptosis signaling pathway.

GPER agonist G1 suppresses neuronal apoptosis mediated by endoplasmic reticulum stress after cerebral ischemia/reperfusion injury

Studies have confirmed a strong association between activation of the endoplasmic reticulum stress pathway and cerebral ischemia/reperfusion(I/R) injury.In this study,three key proteins in the endoplasmic reticulum stress pathway(glucose-regulated protein 78,caspase-12,and C/EBP homologous protein) were selected to examine the potential mechanism of endoplasmic reticulum stress in the neuroprotective effect of G protein-coupled estrogen receptor.Female Sprague-Dawley rats received ovariectomy(OVX),and then cerebral I/R rat models(OVX+ I/R) were established by middle cerebral artery occlusion.Immediately after I/R,rat models were injected with 100 μg/kg E2(OVX + I/R +E2),or 100 μg/kg G protein-coupled estrogen receptor agonist G1(OVX + I/R + G1) in the lateral ventricle.Longa scoring was used to detect neurobehavioral changes in each group.Infarct volumes were measured by 2,3,5-triphenyltetrazolium chloride staining.Morphological changes in neurons were observed by Nissl staining.Terminal dexynucleotidyl transferase-mediated nick end-labeling staining revealed that compared with the OVX + I/R group,neurological function was remarkably improved,infarct volume was reduced,number of normal Nissl bodies was dramatically increased,and number of apoptotic neurons in the hippocampus was decreased after E2 and G1 intervention.To detect the expression and distribution of endoplasmic reticulum stress-related proteins in the endoplasmic reticulum,caspase-12 distribution and expression were detected by immunofluorescence,and mRNA and protein levels of glucose-regulated protein 78,caspase-12,and C/EBP homologous protein were determined by polymerase chain reaction and western blot assay.The results showed that compared with the OVX+ I/R group,E2 and G1 treatment obviously decreased mRNA and protein expression levels of glucose-regulated protein 78,C/EBP homologous protein,and caspase-12.However,the G protein-coupled estrogen receptor antagonist G15(OVX + I/R + E2 + G15) could eliminate the effect of E2 on cerebral I/R injury.These results confirm that E2 and G protein-coupled estrogen receptor can inhibit the expression of endoplasmic reticulum stress-related proteins and neuronal apoptosis in the hippocampus,thereby improving dysfunction caused by cerebral I/R injury.Every experimental protocol was approved by the Institutional Ethics Review Board at the First Affiliated Hospital of Shihezi University School of Medicine,China(approval No.SHZ A2017-171) on February 27,2017.

Role of endoplasmic reticulum stress in the loss of retinal ganglion cells in diabetic retinopathy

Endoplasmic reticulum stress is closely involved in the early stage of diabetic retinopathy. In the present study, a streptozotocin-induced diabetic animal model was given an intraperitoneal injection of tauroursodeoxycholic acid. Results from immunofluorescent co-localization experiments showed that both caspase-12 protein and c-Jun N-terminal kinase 1 phosphorylation levels significantly in- creased, which was associated with retinal ganglion cell death in diabetic retinas. The C/ERB ho- mologous protein pathway directly contributed to glial reactivity, and was subsequently responsible for neuronal loss and vascular abnormalities in diabetic retinopathy. Our experimental findings in- dicate that endoplasmic reticulum stress plays an important role in diabetes-induced retinal neu- ronal loss and vascular abnormalities, and that inhibiting the activation of the endoplasmic reticulum stress pathway provides effective protection against diabetic retinopathy.

Neuroprotective role of edaravone and the effects of endoplasmic reticulum stress in an adult rat model of focal cerebral ischemia/reperfusion

BACKGROUND： Endoplasmic reticulum （ER） stress impairs ER functions and leads to the accumulation of unfolded or misfolded proteins in the ER lumen. ER stress-induced cell death plays an important role in cerebral ischemia. Edaravon （3-methyl-1-phenyl-2-pyrazolin-5-one） is a potent and novel scavenger of free radicals that inhibit delayed neuronal death, as demonstrated by in vitro and animal studies. However, its effect on ER stress and induced neuronal apoptosis in a rat model of brief middle cerebral artery occlusion remains unclear. OBJECTIVE： To explore the effects of edaravone on the expression of ER stress-related factors and neuronal apoptosis, based on the hypothesis that edaravone influences ER stress in a rat model of cerebral ischemia/reperfusion. DESIGN, TIME AND SETTING： A randomized, controlled, animal study was performed at the Laboratory of Department of Neurology, Xiangya Hospital and the Department of Laboratory Animals, Xiangya Medical College, Central South University in China from June 2005 to May 2006. MATERIALS： Edaravone was purchased from Simcere Pharmaceutical Group, China. METHODS： A total of 216 adult, male, Sprague Dawley rats were randomly assigned to sham-surgery, model and edaravone groups, with 72 rats in each group, Brief middle cerebral artery occlusion was established in the model and edaravone groups. In addition, the edaravone group rats were injected with 3 mg/kg edaravone through the tail vein. MAIN OUTCOME MEASURES： RNA-dependent protein kinase-like endoplasmic reticulum eukaryotic translation initiation factor 2a kinase （PERK） and C/EBP homology protein （CHOP） mRNA expression in the ischemic parietal cortex was determined by reverse transcriptionpolymerase chain reaction; phosphorylated PERK and CHOP protein expression was detected by immunohistochemistry; neuronal apoptosis was detected by TdT-mediated-dUTP nick end labeling. RESULTS： Neurological deficit scores were significantly reduced in the edaravone group compared to the model group at 12, 24, and 72 hours following reperfusion （P〈 0.05）. In addition, PERK and CHOP mRNA as well as phosphorylated PERK and CHOP protein expression were significantly reduced in the edaravone group compared to the model group at 1,3, and 6 hours following reperfusion （P 〈 0.05, P 〈 0.01）. CHOP mRNA expression was decreased in the edaravone group compared to the model group at 3, 6, 12, and 24 hours following reperfusion （P〈 0.01）, while CHOP protein expression was less than the model group at 6, 12, and 24 hours following reperfusion （P 〈 0.05）. CONCLUSION： Edaravone treatment resulted in decreased PERK and CHOP expression following ischemia/reperfusion, as well as reduced neuronal apoptosis. Edaravone exhibited a neuroprotective role by inhibiting endoplasmic reticulum stress.

Neuroprotective effects of Activin A on endoplasmic reticulum stress-mediated apoptotic and autophagic PC12 cell death

Activin A, a member of the transforming growth factor-beta superfamily, plays a neuroprotective role in multiple neurological diseases. Endoplasmic reticulum（ER） stress-mediated apoptotic and autophagic cell death is implicated in a wide range of diseases, including cerebral ischemia and neurodegenerative diseases. Thapsigargin was used to induce PC12 cell death, and Activin A was used for intervention. Our results showed that Activin A significantly inhibited morphological changes in thapsigargin-induced apoptotic cells, and the expression of apoptosis-associated proteins [cleaved-caspase-12, C/EBP homologous protein（CHOP） and cleaved-caspase-3] and biomarkers of autophagy（Beclin-1 and light chain 3）, and downregulated the expression of thapsigargin-induced ER stress-associated proteins [inositol requiring enzyme-1（IRE1）, tumor necrosis factor receptor-associated factor 2（TRAF2）, apoptosis signal-regulating kinase 1（ASK1）, c-Jun N-terminal kinase（JNK） and p38]. The inhibition of thapsigargin-induced cell death was concentration-dependent. These findings suggest that administration of Activin A protects PC12 cells against ER stress-mediated apoptotic and autophagic cell death by inhibiting the activation of the IRE1-TRAF2-ASK1-JNK/p38 cascade.

流体剪应力对人脐静脉内皮细胞葡萄糖调节蛋白78和C/EBP同源蛋白表达的影响

目的探索流体剪应力对人脐静脉内皮细胞(HUVECs)葡萄糖调节蛋白78(GRP78)和C/EBP同源蛋白(CHOP)表达的影响。方法以HUVECs作为实验细胞,设计并构建流体动力学模拟实验系统,控制流体动力学模拟实验系统中灌流液的流速,以实现对实验细胞施加不同的流体剪应力。按实验细胞在实验系统中所承受的不同流体剪应力,将实验细胞分为低剪应力组(A组;0.4 Pa)、中剪应力组(B组;0.8 Pa)和高剪应力组(C组;1.2 Pa)。每组HUVECs包含3个细胞玻片,每个玻片经实验系统灌流液反复循环流经12 h。采用蛋白质印迹法对各组细胞中GRP78和CHOP蛋白水平进行检测,采用实时荧光定量逆转录聚合酶链反应技术测定各组细胞GRP78和CHOP蛋白及其信使RNA(mRNA)相对水平。应用GraphPad Prism 8.0软件对数据进行统计学分析。结果(1)A、B、C组HUVECs中GRP78蛋白相对表达水平分别为1.33±0.46、0.93±0.34、0.64±0.30;多组间比较差异有统计学意义(F=36.17,P<0.05)。A组GRP78蛋白相对表达水平高于B组、C组(均P<0.01),B组GRP78蛋白相对表达水平高于C组(P=0.0013)。3组HUVECs中CHOP蛋白相对表达水平分别为:A组1.29±0.38,B组0.90±0.34,C组0.59±0.29;多组间比较差异有统计学意义(F=41.27,P<0.05)。A组CHOP蛋白相对表达水平高于B组、C组(均P<0.01),B组CHOP蛋白相对表达水平高于C组(P=0.0004)。(2)A、B、C组HUVECs中GRP78 mRNA相对表达水平分别为18.3±3.4、11.3±1.8、5.4±2.2;多组间比较差异有统计学意义(F=189.20,P<0.05)。A组GRP78 mRNA相对表达水平高于B组、C组(均P<0.01),B组GRP78 mRNA相对表达水平高于C组(P<0.01)。3组HUVECs中CHOP mRNA相对表达水平分别为:A组20.4±3.8,B组14.2±2.1,C组7.8±1.3;多组间比较差异有统计学意义(F=171.80,P<0.05)。A组CHOP mRNA相对表达水平高于B组、C组(均P<0.01),B组CHOP mRNA相对表达水平高于C组(P<0.01)。结论低流体剪应力可能增加HUVECs中GRP78、CHOP的蛋白及其mRNA表达水平。

Secoemestrin C抗肝癌作用机制研究

目的 通过验证secoemestrin C(Sec C)对肝癌细胞增殖的影响,观察细胞内脂质过氧化水平,检测内质网应激(ERS)相关信号通路变化,进而揭示SecC在抑制肝癌增殖过程中的作用机制,为针对肝癌的药物研发提供新的思路。方法 四甲基偶氮唑蓝(MTT)实验和平板克隆法测定Sec C对Hep G2和BEL7404细胞增殖的影响;流式细胞术测定SecC对肝癌细胞脂质过氧化和凋亡的影响;Westernblot法探究SecC对肝癌细胞内质网应激和细胞凋亡相关蛋白表达的影响。结果 SecC以剂量依赖性方式抑制Hep G2和BEL7404细胞增殖,IC50分别为1.556和3.489μmol/L;平板克隆实验结果显示,1μmol/L Sec C处理7 d后,肝癌细胞的克隆数目和大小显著降低,表明SecC显著抑制肝癌细胞增殖且呈现浓度依赖。流式结果显示,与对照相比,Sec C处理后,Bodipy的阳性率增加且呈现浓度依赖,表明SecC促进肝癌细胞内脂质过氧化,且流式结果显示SecC以剂量依赖性的方式诱导肝癌细胞凋亡。Western blot结果表明,SecC会引起内质网应激相关蛋白免疫球蛋白结合蛋白、1型内质网转膜蛋白激酶、活化转录因子4和磷酸化的真核生物起始因子2表达增多,其作用呈现浓度依赖性;同时Western blot结果显示,凋亡相关蛋白多聚腺嘌呤二核苷酸核糖聚合酶的表达显著降低,以及凋亡剪切产物聚腺苷二磷酸核糖聚合酶、活化的含半胱氨酸的天冬氨酸蛋白水解酶3和活化的含半胱氨酸的天冬氨酸蛋白水解酶7的表达显著增多,其作用呈现浓度依赖性。结论 Sec C能够显著抑制肝癌细胞的增殖,其作用机制可能通过诱导细胞内质网应激导致细胞凋亡。

C/EBPβ在人正常肝细胞和肝癌细胞系中的差异表达及其与内质网应激相关细胞死亡的关系

目的观察转录因子C/EBPβ在人永生化正常肝细胞和肝癌细胞系中的表达差异性,探讨其与肝癌细胞内质网应激介导的细胞死亡的相关性。方法培养人永生化正常肝细胞系HHL5、HL7702和肝癌SMMC7721、Bel7402、HepG2、Hep3B细胞系;Hep3B细胞用衣霉素诱导内质网应激细胞模型;倒置光学显微镜下观察细胞形态;MTT法检测细胞生长抑制率;Hoechst 33258染色后荧光显微镜下检测细胞凋亡;RT-PCR和Western blotting技术分别检测mRNA和蛋白的表达水平。结果 C/EBPβ的mRNA和蛋白亚型C/EBPβ-1在4种人肝癌细胞系和永生化正常肝细胞系中均有表达,但C/EBPβ-3仅在人永生化正常肝细胞系中表达,在肝癌细胞系中不表达;衣霉素能够上调人肝癌Hep3B细胞C/EBPβ的mRNA和蛋白表达水平,明显上调C/EBPβ-3的表达水平;衣霉素能够诱导人肝癌Hep3B细胞内质网应激介导的细胞死亡,表现为细胞凋亡和类凋亡两种方式。结论 C/EBPβ的蛋白亚型C/EBPβ-3在人永生化正常肝细胞中表达而在肝癌细胞中不表达;C/EBPβ参与内质网应激介导的人肝癌细胞死亡。

内质网应激通过C/EBP同源蛋白调控死亡受体5对肝星状细胞凋亡的影响

目的:研究内质网应激(ERS)与肿瘤坏死因子相关凋亡诱导配体(TRAIL)对肝星状细胞凋亡的影响以及在凋亡过程中二者相互关系。方法:以大鼠肝星状细胞HSC-T6为研究对象,使用毒胡萝卜素作为内质网应激诱导剂,熊去氧胆酸作为内质网应激抑制剂,SP600125作为c-Jun氨基末端激酶(JNK)抑制剂,将HSC-T6分成正常组、DMSO组、TRAIL组、毒胡萝卜素组、熊去氧胆酸组、siCHOP组及SP600125组。利用流式法检测毒胡萝卜素诱导HSC-T6凋亡程度;应用小分子RNA干扰技术沉默CHOP基因;免疫组化法检测Caspase-8表达;RT-PCR与Western blot法检测ERS标志性蛋白C/EBP同源蛋白(CHOP)及肿瘤坏死因子相关凋亡诱导配体(TNF-related apoptosis inducing ligand,TRAIL)受体死亡受体5(DR5)的表达。结果:随着毒胡萝卜素浓度增加(1μmol/L,2μmol/L,4μmol/L,8μmol/L,16μmol/L),可诱导HSC-T6发生不同程度的凋亡。RT-PCR与Western blot结果表明ERS标志性蛋白CHOP可诱导TRAIL受体DR5及Caspase-8表达上调;同时,siCHOP及JNK抑制剂SP600125的应用,均可使HSC细胞中DR5及下游Caspase-8的表达随之减少。结论:CHOP和JNK可能是调节DR5表达的潜在因子,两者在诱导肝星状细胞凋亡的过程中发挥着重要的作用。

Secoemestrin C抑制肺腺癌细胞增殖和诱导凋亡的作用机制研究

目的通过检测Secoemestrin C(Sec C)对肺腺癌细胞增殖的影响,观察内质网应激相关基因及细胞信号通路的变化,揭示Sec C在抑制肺腺癌增殖过程的作用机制,为肺腺癌治疗药物的开发提供新的思路。方法以不同浓度梯度的Sec C处理肺腺癌A549和H1299细胞,观察细胞的形态学变化和生长增殖情况,四甲基偶氮唑蓝(MTT)法和平板克隆实验测定Sec C对A549和H1299细胞增殖的影响;5-乙炔基-2'脱氧尿嘧啶核苷(EdU)检测法测定Sec C对A549和H1299细胞DNA复制的影响;Western blot法探究Sec C对A549和H1299细胞内质网应激和细胞凋亡相关蛋白的表达变化。结果光镜下观察发现Sec C对A549和H1299具有杀伤作用,IC_(50)分别为2.164和2.341μmol/L;克隆形成实验结果表明Sec C能够抑制A549和H1299细胞的增殖能力,且呈浓度依赖;EdU实验结果证明Sec C能够显著抑制DNA复制。Western blot结果表明,Sec C会引起内质网应激相关蛋白免疫球蛋白结合蛋白(BiP)、活化转录因子4(ATF4)、磷酸化的真核生物起始因子2(p-eIF2)、1型内质网转膜蛋白激酶(IRE1α)表达增多,其作用呈时间和浓度依赖性;Sec C可诱导细胞凋亡,使半胱氨酸天冬氨酸酶3(caspase3)、半胱氨酸天冬氨酸酶7(caspase7)、多聚腺嘌呤二核苷酸核糖聚合(PARP)的表达显著降低,以及活化的含半胱氨酸的天冬氨酸蛋白水解酶3(cleaved-caspase3)、活化的含半胱氨酸的天冬氨酸蛋白水解酶7(cleaved-caspase7)、凋亡剪切产物聚腺苷二磷酸核糖聚合酶(cleaved-PARP)的表达显著增多,其作用呈时间和浓度依赖性。结论Sec C能明显抑制肺腺癌细胞的增殖活性,其作用机制可能通过内质网应激诱导细胞凋亡。

橙皮素通过激活内质网应激C/EBP同源蛋白诱导人神经胶质瘤细胞凋亡实验研究

目的:分析橙皮素(HES)对人神经胶质瘤细胞凋亡的影响及相关分子机制。方法:将人神经胶质瘤U251细胞分为100μmol/L橙皮素组、200μmol/L橙皮素组、400μmol/L橙皮素组、600μmol/L橙皮素组、800μmol/L橙皮素组、阴性对照组、4-苯基丁酸(4-PBA)组、橙皮素加4-苯基丁酸组、转染对照组、C/EBP同源蛋白(CHOP)低表达组、橙皮素加转染对照组、橙皮素加CHOP低表达组。采用CCK-8法检测细胞增殖活性,Hoechst 33258染色法和Annexin V/PI双染法检测细胞凋亡率,qRT-PCR法检测CHOP mRNA表达水平,Western blot法检测细胞凋亡蛋白和内质网应激(ERS)蛋白表达水平。结果:HES对胶质瘤U251细胞的增殖抑制作用呈剂量和时间依赖性,与阴性对照组比较,HES可诱导U251细胞凋亡,上调Caspase-3、剪切Caspase-3、多聚ADP核糖聚合酶(PARP)、剪切PARP蛋白以及CHOP、ERS蛋白分子伴侣葡萄糖调节蛋白78、X盒结合蛋白1(XBP-1)、激活转录因子6蛋白表达量和磷酸化真核起始因子2α/eIF2α。4-PBA可逆转HES引起的ERS蛋白和凋亡蛋白表达变化;干扰CHOP表达可逆转HES对凋亡蛋白的调控作用。结论:HES通过介导ERS途径CHOP诱导胶质瘤细胞凋亡。

Silencing the gene encoding C/EBP homologous protein lessens acute brain injury following ischemia/reperfusion

C/EBP homologous protein, an important transcription factor during endoplasmic reticulum stress, participates in cell apoptosis mediated by endoplasmic reticulum stress. Previous studies have shown that C/EBP homologous protein mediates nerve injury during Alzheimer＇s disease, subarachnoid hemorrhage and spinal cord trauma. In this study, we introduced C/EBP homologous protein short hairpin RNA into the brains of ischemia/reperfusion rat models via injection of lentiviral vector through the left lateral ventricle. Silencing C/EBP homologous protein gene expression significantly reduced cerebral infarction volume, decreased water content and tumor necrosis factor-α and interleukin-1β mRNA expression in brain tissues following infarction, diminished the number of TUNEL-positive cells in the infarct region, decreased caspase-3 protein content and increased Bcl-2 protein content. These results suggest that silencing C/EBP homologous protein lessens cell apoptosis and inflammatory reactions, thereby protecting nerves.

Valproate reduces retinal ganglion cell apoptosis in rats after optic nerve crush

The retinal ganglion cells of the optic nerve have a limited capacity for self-repair after injury.Valproate is a histone deacetylase inhibitor and multitarget drug,which has been demonstrated to protect retinal neurons.In this study,we established rat models of optic nerve-crush injury and injected valproate into the vitreous cavity immediately after modeling.We evaluated changes in the ultrastructure morphology of the endoplasmic reticulum of retinal ganglion cells over time via transmission electron microscope.Immunohistochemistry and western blot assay revealed that valproate upregulated the expression of the endoplasmic reticulum stress marker glucose-regulated protein 78 and downregulated the expression of transcription factor C/EBP homologous protein,phosphorylated eukaryotic translation initiation factor 2α,and caspase-12 in the endoplasmic reticulum of retinal ganglion cells.These findings suggest that valproate reduces apoptosis of retinal ganglion cells in the rat after optic nerve-crush injury by attenuating phosphorylated eukaryotic translation initiation factor 2α-C/EBP homologous protein signaling and caspase-12 activation during endoplasmic reticulum stress.These findings represent a newly discovered mechanism that regulates how valproate protects neurons.

靶向GPCRs的药物筛选方法

G蛋白偶联受体(G-protein coupled receptor,GPCR)是一类7-次跨膜蛋白,参与介导多种重要的生理功能,是畅销药物中占比最高和最成功的靶点之一。几十年来,靶向GPCR的药物研发一直是学术界和制药界的热门焦点,这些已发现并批准使用的GPCR药物为多种人类疾病的治疗提供了可能性,包括癌症、病毒感染、炎症性疾病和代谢性疾病等,例如艾塞那肽、利拉鲁肽、利西拉肽和阿比鲁肽等靶向胰高血糖素样肽1受体的肽类药物已用于2型糖尿病的治疗[1];靶向钙敏感受体的变构调节剂西那卡塞已批准用于甲状旁腺功能亢进治疗[2]。

Study on C/EBPα and FOXC2 expression in colon cancer lesions andpreliminary analysis of their downstream target genes

Objective: To study the C/EBPα and FOXC2 expression in colon cancer lesions and explore their downstream target genes. Methods: Colon cancer specimens surgically removed in Weinan Central Hospital between March 2014 and February 2017 were selected, and adjacent tissue specimens more than 5cm from tumor lesion margin were selected as control. The RNA in the clinical specimens was extracted and the mRNA expression of C/EBPα, FOXC2 as well as endoplasmic reticulum stress molecules and epithelial-mesenchymal transition molecules were determined. Results: C/EBPα, Bax, Bak, GRP78, PERK and ATF6 mRNA expression levels in colon cancer specimens were significantly lower than those in adjacent samples while FOXC2, FAK, SphK1, N-cadherin and Vimentin mRNA expression levels were significantly higher than those in adjacent specimens;Bax, Bak, GRP78, PERK and ATF6 mRNA expression levels in colon cancer specimens with high C/EBPα expression were significantly higher than those in colon cancer specimens with low C/EBPα expression;FAK, SphK1, N-cadherin and Vimentin mRNA expression levels in colon cancer specimens with high FOXC2 expression were significantly higher than those in colon cancer specimens with low FOXC2 expression. Conclusion: Lowly expressed C/EBPα and highly expressed FOXC2 in colon cancer lesions can inhibit the apoptosis mediated by endoplasmic reticulum stress and promote the cell invasion mediated by epithelial-mesenchymal transition.

Role of endoplasmic reticulum stress protein C / EBP homologous protein-10 in ischemia and hypoxia induced human aortic endothelial cells injury

Objective To investigate the expression of endoplasmic reticulum stress(ESR)marker C/EBP homologous protein-10(CHOP-10)in the human aortic endothelial cells(HAEC)under the ischemia and hypoxia stress and to study the effects of atorvastatin on the process.Methods The cultured HAEC were divided into normal control group,ischemia/hypoxia model group。

抑制内质网应激关键信号通路蛋白eIF2α去磷酸化与肝细胞增殖及c-Met表达的关系

目的探讨抑制内质网应激(ERS)关键信号通路蛋白真核细胞起始因子2α(eIF2α)去磷酸化与肝细胞增殖及促肝细胞生长因子受体(c-Met)表达的关系。方法将人肝细胞株L02分为对照组和胡萝卜素(TG)组,TG组加入含内质网钙平衡抑制剂毒胡萝卜素(TG)的RPMI1640培养基,诱导构建细胞ERS模型;对照组仅加入RPMI1640培养基,培养48 h。采用CCK-8检测细胞活力,Western blotting法检测细胞ESR相关蛋白p-eIF2α、ERAD通路相关蛋白HRD1、XBP1以及c-Met蛋白表达。另取L02细胞,分为TG组和Salubrinal+TG组,Salubrinal+TG组给予eIF2α去磷酸化抑制剂Salubrinal预处理2 h,然后加入1μmol/L TG培养48 h;TG组加入1μmol/L TG培养48 h。采用CCK-8检测细胞活力,Western blotting法检测细胞eIF2α、p-eIF2α、HRD1、XBP1、c-Met蛋白表达。结果与对照组相比,TG组p-eIF2α、HRD1表达水平升高,TG组ERS相关蛋白p-eIF2α表达上调,ERAD通路激活,表明ERS细胞模型构建成功。TG组细胞增殖活力和c-Met蛋白表达水平均低于对照组(P均<0.05)。与TG组相比,Salubrinal+TG组细胞XBP1、HRD1蛋白表达降低,细胞增殖活力和c-Met蛋白表达升高(P均<0.05)。结论在肝细胞ERS中,抑制eIF2α去磷酸化能够部分恢复肝细胞细胞增殖活力及c-Met表达,促进细胞存活。

低剂量电离辐射诱导小鼠睾丸细胞内质网应激及PERK-CHOP信号通路的激活

目的:探讨低剂量电离辐射与小鼠睾丸细胞内质网应激的发生以及PERK-CHOP通路激活的相关性。方法:健康雄性昆明小鼠随机分成时程-效应(75 mGy照射后0、3、6、12和24 h)和剂量-效应(0、50、75、100和200 mGy照射后12 h)组,每组动物10只。采用H2O2和MDA试剂盒比色法检测其含量;利用实时定量逆转录PCR(quantitative RT-PCR)检测GRP78、PERK和CHOP mRNA;Western印迹和图像分析技术检测GRP78、PERK、磷酸化PERK(phosphorylated PERK,pho-PERK)和CHOP蛋白表达。结果:小鼠经75 mGy全身照射后,睾丸组织中H2O2含量随时间延长而增加,MDA含量在3和6 h稍有降低,而后随时间延长而增加,二者在12和24 h较0 h时显著增加(P<0.05,P<0.01);除了GRP78 mRNA(3和24 h)和蛋白表达(6 h)分别在照射后有降低趋势外,GRP78(12 h)、PERK(3、6、12和24 h)和CHOP(12和24 h)的mRNA表达较0 h显著增加(P<0.05,P<0.01),GRP78(12和24 h)、pho-PERK(3、12和24 h)和CHOP(3、6、12和24 h)的蛋白表达也都较0 h显著增加(P<0.05,P<0.01),PERK蛋白表达则无明显变化规律。小鼠经50～200 mGy全身照射后12 h,睾丸组织中H2O2含量在50～100 mGy照射后随剂量增加而增加,200 mGy照射后则稍有降低,MDA含量随剂量增加而增加,而且H2O2含量(75和100 mGy)和MDA含量(75、100和200 mGy)显著高于0 mGy组(P<0.05,P<0.01);除了GRP78mRNA表达在50和200 mGy照射后有降低趋势外,GRP78(75和100 mGy)、PERK(75、100和200 mGy)和CHOP(50、75、100和200 mGy)的mRNA表达都显著高于0 mGy组(P<0.05,P<0.01),GRP78(100和200 mGy)、pho-PERK(50、100和200 mGy)和CHOP(50、75、100和200 mGy)的蛋白表达也都显著高于0 mGy组(P<0.05,P<0.01),而PERK蛋白表达则无明显变化规律。结论:低剂量电离辐射能够诱导小鼠睾丸细胞发生内质网应激,并且激活PERK-CHOP信号通路。