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.

Treatment with β-sitosterol ameliorates the effects of cerebral ischemia/reperfusion injury by suppressing cholesterol overload, endoplasmic reticulum stress, and apoptosis

β-Sitosterol is a type of phytosterol that occurs naturally in plants.Previous studies have shown that it has anti-oxidant,anti-hyperlipidemic,anti-inflammatory,immunomodulatory,and anti-tumor effects,but it is unknown whetherβ-sitosterol treatment reduces the effects of ischemic stroke.Here we found that,in a mouse model of ischemic stroke induced by middle cerebral artery occlusion,β-sitosterol reduced the volume of cerebral infarction and brain edema,reduced neuronal apoptosis in brain tissue,and alleviated neurological dysfunction;moreover,β-sitosterol increased the activity of oxygen-and glucose-deprived cerebral cortex neurons and reduced apoptosis.Further investigation showed that the neuroprotective effects ofβ-sitosterol may be related to inhibition of endoplasmic reticulum stress caused by intracellular cholesterol accumulation after ischemic stroke.In addition,β-sitosterol showed high affinity for NPC1L1,a key transporter of cholesterol,and antagonized its activity.In conclusion,β-sitosterol may help treat ischemic stroke by inhibiting neuronal intracellular cholesterol overload/endoplasmic reticulum stress/apoptosis signaling pathways.

Bushen Yizhi Formula regulates the IRE1αpathway to alleviate endoplasmic reticulum stress in an Alzheimer’s disease rat model

While the Bushen Yizhi Formula can treat Alzheimer’s disease(AD),the yet to be ascertained specific mechanism of action was explored in this work.Methods:Different concentrations of the Bushen Yizhi Formula and amyloid-beta peptide(Aβ)were used to treat rat pheochromocytoma cells(P12)and human neuroblastoma cells(SH-SY5Y).Cell morphological changes were observed to determine the in vitro cell damage.Cell Counting Kit(CCK)-8 assay and flow cytometry were employed to identify cell viability and apoptosis/cell cycle,respectively.Western blotting and immunohistochemistry were employed to measure the expressions of endoplasmic reticulum stress(ERS)-related proteins(GRP78 and CHOP),p-IRE1α,IRE1α,ASK1,p-JNK,JNK,Bax,Bcl-2,XBP-1,and Bim.Fura 2-acetoxymethyl ester(Fura-2/AM)was used to determine the intracellular calcium(Ca^(2+))concentration.Also,an AD model was constructed by injecting Aβinto the CA1 area of the hippocampus in Sprague Dawley rats.AD model rats were gavaged with different concentrations of Bushen Yizhi Formula for 14 consecutive days.The Morris water maze experiment was conducted to test the learning and memory of rats.Hematoxylin&Eosin(H&E)and Terminal-deoxynucleotidyl Transferase(TdT)-mediated dUTP Nick-End Labeling(TUNEL)staining were done to determine histopathological changes in the brain.Results:Bushen Yizhi Formula relieved the Aβ-induced effects including cell injury,decreased viability,increased apoptosis,G0/G1 phase cell cycle arrest,upregulation of GRP78,CHOP,p-IRE1α,p-JNK,Bax,XBP-1 and Bim,as well as down-regulation of Bcl-2.These results were also seen with IRE1αsilencing.While Aβsuppressed the learning and memory abilities of rats,the Bushen Yizhi Formula alleviated these effects of Aβ.Brain nerve cell injury induced by Aβcould also be treated with Bushen Yizhi Formula.Conclusion:Bushen Yizhi Formula could influence ERS through the IRE1αsignaling pathway to achieve its therapeutic effects on AD.

PINK1, Keap1, and Rtnl1 regulate selective clearance of endoplasmic reticulum during development

Selective clearance of organelles,including endoplasmic reticulum(ER)and mitochondria,by autophagy plays an important role in cell health.Here,we describe a developmentally programmed selective ER clearance by autophagy.We show that Parkinson's disease-associated PINK1,as well as Atl,Rtnl1,and Trp1 receptors,regulate ER clearance by autophagy.

Sirtuin 1 alleviates endoplasmic reticulum stress-mediated apoptosis of intestinal epithelial cells in ulcerative colitis

BACKGROUND Sirtuin 1(SIRT1)is a nicotinamide adenine dinucleotide(NAD+)-dependent protein deacetylase that is involved in various diseases,including cancers,metabolic diseases,and inflammation-associated diseases.However,the role of SIRT1 in ulcerative colitis(UC)is still confusing.AIM To investigate the role of SIRT1 in intestinal epithelial cells(IECs)in UC and further explore the underlying mechanisms.METHODS We developed a coculture model using macrophages and Caco-2 cells.After treatment with the SIRT1 activator SRT1720 or inhibitor nicotinamide(NAM),the expression of occludin and zona occludens 1(ZO-1)was assessed by Western blot analysis.Annexin V-APC/7-AAD assays were performed to evaluate Caco-2 apoptosis.Dextran sodium sulfate(DSS)-induced colitis mice were exposed to SRT1720 or NAM for 7 d.Transferase-mediated dUTP nick-end labeling(TUNEL)assays were conducted to assess apoptosis in colon tissues.The expression levels of glucose-regulated protein 78(GRP78),CCAAT/enhancerbinding protein homologous protein(CHOP),caspase-12,caspase-9,and caspase-3 in Caco-2 cells and the colon tissues of treated mice were examined by quantitative real-time PCR and Western blot.RESULTS SRT1720 treatment increased the protein levels of occludin and ZO-1 and inhibited Caco-2 apoptosis,whereas NAM administration caused the opposite effects.DSS-induced colitis mice treated with SRT1720 had a lower disease activity index(P<0.01),histological score(P<0.001),inflammatory cytokine levels(P<0.01),and apoptotic cell rate(P<0.01),while exposure to NAM caused the opposite effects.Moreover,SIRT1 activation reduced the expression levels of GRP78,CHOP,cleaved caspase-12,cleaved caspase-9,and cleaved caspase-3 in Caco-2 cells and the colon tissues of treated mice.CONCLUSION SIRT1 activation reduces apoptosis of IECs via the suppression of endoplasmic reticulum stress-mediated apoptosis-associated molecules CHOP and caspase-12.SIRT1 activation may be a potential therapeutic strategy for UC.

Hydrogen-rich water protects against inflammatory bowel disease in mice by inhibiting endoplasmic reticulum stress and promoting heme oxygenase-1 expression

AIM To investigate the therapeutic effect of hydrogen-rich water(HRW) on inflammatory bowel disease(IBD) and to explore the potential mechanisms involved.METHODS Male mice were randomly divided into the following four groups: control group, in which the mice received equivalent volumes of normal saline(NS) intraperitoneally(ip); dextran sulfate sodium(DSS) group, in which the mice received NS ip(5 m L/kg body weight, twice per day at 8 am and 5 pm) for 7 consecutive days after IBD modeling; DSS + HRW group, in which the mice received HRW(in the same volume as the NS treatment) for 7 consecutive days after IBD modeling; and DSS + HRW + Zn PP group, in which the mice received HRW(in the same volume as the NS treatment) and ZnP P [a heme oxygenase-1(HO-1) inhibitor, 25 mg/kg] for 7 consecutive days after IBD modeling. IBD was induced by feeding DSS to the mice, and blood and colon tissues were collected on the 7th d after IBD modeling to determine clinical symptoms, colonic inflammation and the potential mechanisms involved.RESULTS The DSS + HRW group exhibited significantly attenuated weight loss and a lower extent of disease activity index compared with the DSS group on the 7th d(P < 0.05). HRW exerted protective effects against colon shortening and colonic wall thickening in contrast to the DSS group(P < 0.05). The histological study demonstrated milder inflammation in the DSS + HRW group, which was similar to normal inflammatory levels, and the macroscopic and microcosmic damage scores were lower in this group than in the DSS group(P < 0.05). The oxidative stress parameters, including MDA and MPO in the colon, were significantly decreased in the DSS + HRW group compared with the DSS group(P < 0.05). Simultaneously, the protective indicators, superoxide dismutase and glutathione, were markedly increased with the use of HRW. Inflammatory factors were assessed, and the results showed that the DSS + HRW group exhibited significantly reduced levels of TNF-α, IL-6 and IL-1β compared with the DSS group(P < 0.05). In addition, the pivotal proteins involved in endoplasmic reticulum(ER) stress, including p-e IF2α, ATF4, XBP1 s and CHOP, were dramatically reduced after HRW treatment in contrast to the control group(P < 0.05). Furthermore, HRW treatment markedly up-regulated HO-1 expression, and the use of Zn PP obviously reversed the protective role of HRW. In the DSS + HRW + ZnP P group, colon shortening and colonic wall thickening were significantly aggravated, and the macroscopic damage scores were similar to those of the DSS + HRW group(P < 0.05). The histological study also showed more serious colonic damage that was similar to the DSS group.CONCLUSION HRW has a significant therapeutic potential in IBD by inhibiting inflammatory factors, oxidative stress and ER stress and by up-regulating HO-1 expression.

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.

Effects of natural cerebrolysin on protective proteins and pro-apoptotic molecules in mesenchymal stem cells following beta-amyloid peptide1-40-induced endoplasmic reticulum stress

BACKGROUND： Studies have demonstrated that β-amyloid peptide （Aβ）, a characteristic pathological product of Alzheimer＇s disease （AD）, results in neuronal endoplasmic reticulum stress （ERS）. However, the mechanisms of traditional Chinese medicine against ERS in AD are poorly understood. OBJECTIVE： To measure expression levels of protective proteins （GRP78 and GRP94） of ER molecular partners and pro-apoptotic Caspase-12 ER membrane expression following application of traditional Chinese medicine natural cerebrolysin （NC） to treat Aβ1-40-induced ERS. DESIGN, TIME AND SETTING： A parallel-controlled study was performed at the Institute of Integrated Western and Traditional Chinese Medicine, Shenzhen Hospital of Southern Medical University between September 2006 and November 2008. MATERIALS： Sprague Dawley male rats, 6-8 weeks old, were used to harvest tibial and femoral bone marrow. Isolation and purification of mesenchymal stem cells （MSCs） were established from the whole bone marrow by removing non-adherent cells in primary and passage cultures. Aβ1-40 was provided by Sigma, USA. NC was provided by Shenzhen Institute of Integrated Chinese and Western Medicine, China. NC was predominantly composed of Renshen （Radix Ginseng）, Tianma （Rhizoma Gastrodiae）, and Yinxingye （Ginkgo Leaf） in a proportion of 1 ： 2： 2. Following conventional water extraction technology, an extract （1 ： 20） was prepared. Six adult, male, New Zealand rabbits underwent intragastric administration of NC extract （0.976 g/kg per day） for 1 month to prepare NC-positive serum, and the remaining 6 rabbits received intragastric administration of physiological saline to prepare normal blank serum. METHODS： A total of 500 nmol/L Aβ1-40 was used to establish ERS models of primary cultured MSCs. AD cell models were incubated with different doses of NC-positive serum （2.5%, 5%, and 10%）. MSCs treated with normal blank serum served as normal blank controls. MAIN OUTCOME MEASURES： Reverse transcription-polymerase chain reaction and fluorescent immunocytochemistry were respectively used to measure mRNA and protein expression levels of GRP78, GRP94, and Caspase-12 in MSCs. RESULTS： Following Aβ1-40 exposure, mRNA and protein expression levels of GRP78 and GRP94, as well as Caspase-12, significantly increased （P 〈 0.05）, suggesting successful establishment of ERS models. Following NC-positive serum application, mRNA and protein expression levels of GRP78 and GRP94 in MSCs significantly increased （P 〈 0.05 or P 〈 0.01）. However, mRNA and protein expression levels of Caspase-12 significantly decreased （P 〈 0.05, or P 〈 0.01） compared with the ERS model group. These effects were dose-dependent. CONCLUSION： NC downregulated Caspase-12 expression and upregulated GRP78 and GRP94 expression in MSCs in a dose-dependent manner under the state of Aβ1-40-induced ERS.

1-methyl-4-phenylpyridinium ion induces endoplasmic reticulum stress through glycogen synthase kinase-3 beta activation in PC12 cells

1-methyl-4-phenylpyridinium ion （MPP^＋） induces endoplasmic reticulum stress and activates caspase-12 in PC12 cells, leading to neuronal apoptosis. However, the underlying molecular mechanism remains unknown. The present study investigated the regulatory effects of nerve growth factor （Akt activator） and lithium chloride （glycogen synthase kinase-3β inhibitor） on the endoplasmic reticulum stress signaling pathway. The results revealed that MPP＋ induced expression of Bip and C/EBP homologous protein. The upregulation of Bip and C/EBP homologous protein, as well as the decreased pro-caspase-12 level induced by MPP^＋ were inhibited by pretreatment of the nerve growth factor or lithium chloride. These results suggest that the phosphatidylinositol 3 kinase-Aktglycogen synthase kinase-3β pathway is involved in MPP-induced endoplasmic reticulum stress.

Z α-1 antitrypsin deficiency and the endoplasmic reticulum stress response

The serine proteinase inhibitor α-1 antitrypsin(AAT) is produced principally by the liver at the rate of 2 g/d.It is secreted into the circulation and provides an antiprotease protective screen throughout the body but most importantly in the lung,where it can neutralise the activity of the serine protease neutrophil elastase.Mutations leading to def iciency in AAT are associated with liver and lung disease.The most notable is the Z AAT mutation,which encodes a misfolded variant of the AAT protein in which the glutamic acid at position 342 is replaced by a lysine.More than 95% of all individuals with AAT def iciency carry at least one Z allele.ZAAT protein is not secreted effectively and accumulates intracellularly in the endoplasmic reticulum(ER) of hepatocytes and other AAT-producing cells.This results in a loss of function associated with decreased circulating and intrapulmonary levels of AAT.However,the misfolded protein acquires a toxic gain of function that impacts on the ER.A major function of the ER is to ensure correct protein folding.ZAAT interferes with this function and promotes ER stress responses and inflammation.Here the signalling pathways activated during ER stress in response to accumulation of ZAAT are described and therapeutic strategies that can potentially relieve ER stress are discussed.

Thrombospondin 1 Promotes Endoplasmic Reticulum Stress and Apoptosis in HK-2 Cells by Upregulating ATF6-CHOP

Objective The goal of this study is to investigate the role and mechanism of endoplasmic reticulum stress and apoptosis regulated by thrombospondin 1(TSP1)in human renal tubular epithelial cells(HK-2 cells).Methods HK-2 cells were exposed to high concentrations of glucose(HG).The endoplasmic reticulum stress inhibitor 4-phenylbutyric acid(4-PBA)was administered by transfecting TSP1 or an empty vector to explore the mechanism of the endoplasmic reticulum response regulated by TSP1 and stress in renal cell apoptosis.The effects of TSP1 and 4-PBA on the proliferation and apoptosis of HK-2 cells under HG conditions were assessed using Cell counting kit-8 and flow cytometry.Western blotting was used to detect the apoptosis-and endoplasmic reticulum stress-related protein expression regulated by TSP1 and 4-PBA.Results HG treatment induced high cell apoptosis,abundantly expressed TSP1 level and restrained viability in HK-2 cells.Overexpression of TSP1 significantly inhibited the proliferation of and facilitated apoptosis of HK-2 cells under HG conditions.Administration of endoplasmic reticulum stress inhibitor 4-PBA after overexpression of TSP1 antagonized the inhibitory proliferation and promoted apoptosis rate in HG-triggered HK-2 cells induced by TSP1 overexpression.4-PBA treatment significantly hindered the expression of endoplasmic reticulum stress markers,such as PERK,ATF4,ATF6,p-eIF2α,IRE1,CHOP and XBP1,suggesting that the administration of 4-PBA was successful.Conclusion Overexpression of TSP1 activated endoplasmic reticulum stress by regulating the ATF6-CHOP axis.TSP1 restrained cell proliferation,and promoted apoptosis and endoplasmic reticulum stress by activating the ATF6-CHOP axis.

Resveratrol reduces brain injury after subarachnoid hemorrhage by inhibiting oxidative stress and endoplasmic reticulum stress

Previous studies have shown that resveratrol,a bioactive substance found in many plants,can reduce early brain injury after subarachnoid hemorrhage,but how it acts is still unclear.This study explored the mechanism using the experimental subarachnoid hemorrhage rat model established by injecting autologous blood into the cerebellomedullary cistern.Rat models were treated with an intraperitoneal injection of 60 mg/kg resveratrol 2,6,24 and 46 hours after injury.At 48 hours after injury,their neurological function was assessed using a modified Garcia score.Brain edema was measured by the wet-dry method.Neuronal apoptosis in the prefrontal cortex was detected by terminal deoxyribonucleotidyl transferase-mediated biotin-16-dUTP nick-end labeling assay.Levels of reactive oxygen species and malondialdehyde in the prefrontal cortex were determined by colorimetry.CHOP,glucose-regulated protein 78,nuclear factor-erythroid2-related factor 2 and heme oxygenase-1 mRNA expression levels in the prefrontal cortex were measured by reverse transcription polymerase chain reaction.Tumor necrosis factor-alpha content in the prefrontal cortex was detected by enzyme linked immunosorbent assay.Immunohistochemical staining was used to detect the number of positive cells of nuclear factor-erythroid 2-related factor 2,heme oxygenase 1,glucose-regulated protein 78,CHOP and glial fibrillary acidic protein.Western blot assay was utilized to analyze the expression levels of nuclear factor-erythroid 2-related factor 2,heme oxygenase 1,glucose-regulated protein 78 and CHOP protein expression levels in the prefrontal cortex.The results showed that resveratrol treatment markedly alleviated neurological deficits and brain edema in experimental subarachnoid hemorrhage rats,and reduced neuronal apoptosis in the prefrontal cortex.Resveratrol reduced the levels of reactive oxygen species and malondialdehyde,and increased the expression of nuclear factor-erythroid 2-related factor 2,heme oxygenase-1 mRNA and protein in the prefrontal cortex.Resveratrol decreased glucose-regulated protein 78,CHOP mRNA and protein expression and tumor necrosis factor-alpha level.It also activated astrocytes.The results suggest that resveratrol exerted neuroprotective effect on subarachnoid hemorrhage by reducing oxidative damage,endoplasmic reticulum stress and neuroinflammation.The study was approved by the Animals Ethics Committee of Shandong University,China on February 22,2016(approval No.LL-201602022).

EDEM1通过稳定ATF6促进颅内动脉瘤发展的作用研究

目的:探究内质网降解增强α-甘露糖苷酶样蛋白1(EDEM1)和激活转录因子6(ATF6)在颅内动脉瘤(IA)发展中的作用。方法:纳入40例颅内动脉瘤患者和40例健康受试者,收集两组人群血清,通过ELISA检测ATF6水平。根据Hunt-Hess分级和Fisher分级将IA患者分为:轻度疾病组、中度疾病组和重度疾病组,比较3组患者血清中ATF6水平。将si-EDEM1和si-NC转染至HCAECs细胞,分组为si-EDEM1组和si-NC组,采用western blot检测两组细胞EDEM1和ATF6蛋白表达水平,CCK-8法检测两组细胞增殖水平。通过免疫共沉淀检测EDEM1和ATF6的相互作用情况。将si-ATF6和si-NC转染至HCAECs细胞,分组为si-ATF6组和si-NC组,采用Western blot检测两组细胞EDEM1和ATF6蛋白表达水平,CCK-8法检测两组细胞增殖水平。结果:与健康受试者相比,颅内动脉瘤患者血清中ATF6水平升高(P<0.05)。ATF6水平在轻度疾病组、中度疾病组和重度疾病组中依次递增(P<0.05)。与si-NC组相比,si-EDEM1组细胞EDEM1和ATF6蛋白表达水平降低,细胞增殖水平降低(P<0.05)。EDEM1和ATF6存在相互作用。与si-NC组相比,si-ATF6组细胞ATF6蛋白表达水平降低,细胞增殖水平降低(P<0.05)。结论:EDEM1通过稳定ATF6促进动脉内皮细胞增殖,与IA的恶化相关。

肺癌组织中ERO1L、TNFRSF4的表达与患者免疫功能、炎症反应因子及预后的关系

目的探究肺癌组织中内质网氧化物蛋白(ERO1L)、肿瘤坏死因子受体4(TNFRSF4)的表达与肺癌患者免疫功能、炎症反应因子及其预后的关系。方法选取2018年7月~2020年7月于本院进行手术治疗的108例肺癌患者,收集术中留取的癌组织和癌旁组织标本。采用qRT-PCR检测ERO1L和TNFRSF4的mRNA相对表达量;使用免疫组织化学法检测ERO1L和TNFRSF4蛋白表达情况,分析二者表达水平与患者临床病理特征的关系,采用Kaplan-Meier法分析ERO1L、TNFRSF4蛋白表达水平与患者预后的关系。肺癌患者预后生存率的影响因素采用Cox多因素分析。结果肺癌患者癌组织中ERO1L mRNA表达水平显著高于癌旁组织,TNFRSF4 mRNA表达水平显著低于癌旁组织(P<0.05);肺癌组织中ERO1L蛋白高表达率显著高于癌旁组织,TNFRSF4蛋白高表达率显著低于癌旁组织(P<0.05)。ERO1L蛋白高表达组患者CD3^(+)、CD4^(+)显著低于低表达组(P<0.05),IL-1β、IL-6、TNF-α显著高于低表达组(P<0.05);TNFRSF4蛋白高表达组患者CD3^(+)、CD4^(+)显著高于低表达组,IL-1β、IL-6、TNF-α显著低于低表达组(P<0.05)。ERO1L高表达组患者3年累积生存率显著低于低表达组(Log rankχ^(2)=6.100,P=0.014),TNFRSF4高表达组患者3年累积生存率显著高于低表达组(Log rankχ^(2)=11.296,P=0.001)。肺癌组织的低分化、淋巴结转移、TNM分期为Ⅲ-Ⅳ期、ERO1L高表达、TNFRSF4低表达是影响患者生存率的危险因素。结论肺癌组织中ERO1L、TNFRSF4表达与患者免疫功能、炎症因子以及预后具有一定关系。

Axon injury induced endoplasmic reticulum stress and neurodegeneration

Injury to central nervous system axons is a common early characteristic of neurodegenerative diseases. Depending on its location and the type of neuron, axon injury often leads to axon degeneration, retrograde neuronal cell death and progressive permanent loss of vital neuronal functions. Although these sequential events are clearly connected, ample evidence indicates that neuronal soma and axon degenerations are active autonomous processes with distinct molecular mechanisms. By exploiting the anatomical and techni- cal advantages of the retinal ganglion cell （RGC）/optic nerve （ON） system, we demonstrated that inhibition of the PERK-eIF2a-CHOP pathway and activation of the X-box binding protein 1 pathway synergistically protect RGC soma and axon, and preserve visual function, in both acute ON traumatic injury and chronic glaucomatous neuropathy. The autonomous endoplasmic reticulum （ER） stress pathway in neurons has been implicated in several other neurodegenerative diseases. In addition to the emerging role of ER mor- phology in axon maintenance, we propose that ER stress is a common upstream signal for disturbances in axon integrity, and that it leads to a retrograde signal that can subsequently induce neuronal soma death. Therefore manipulation of the ER stress pathway may be a key step toward developing the effective neuro- protectants that are greatly needed in the clinic.

下调XBP1s通过抑制ITPR介导的线粒体功能障碍改善肾小管上皮细胞缺氧/复氧损伤

目的 探讨剪接型X盒结合蛋白1(XBP1s)对小鼠肾小管上皮细胞缺氧/复氧(H/R)损伤的影响及其作用机制。方法 将小鼠肾小管上皮细胞分为腺病毒阴性对照组(Ad-shNC组)、靶向沉默XBP1s腺病毒组(Ad-shXBP1s组)、Ad-shNC+H/R组、Ad-shXBP1s+H/R组。检测各组细胞凋亡水平、线粒体活性氧活性、线粒体膜电位及线粒体钙离子水平。使用染色质免疫共沉淀测序(ChIP-seq)分析XBP1s调控肌醇1,4,5-三磷酸受体(ITPR)家族的结合位点。检测各组XBP1s和ITPR家族信使RNA(mRNA)和蛋白表达水平。结果 与AdshNC组比较,Ad-shNC+H/R组细胞凋亡水平更高,线粒体活性氧水平升高,线粒体膜电位降低,线粒体钙离子水平升高;与Ad-shNC+H/R组比较,Ad-shXBP1s+H/R组细胞凋亡水平较低,线粒体活性氧水平下降,线粒体膜电位升高,线粒体钙离子水平降低(均为P<0.05)。与Ad-shNC组比较,Ad-shXBP1s组XBP1s、ITPR1、ITPR2和ITPR3 mRNA和蛋白相对表达量降低(均为P<0.05)。与Ad-shNC组相比,Ad-shNC+H/R组XBP1s、ITPR1、ITPR2和ITPR3蛋白相对表达量升高;与Ad-shNC+H/R组相比,Ad-shXBP1s+H/R组XBP1s、ITPR1、ITPR2和ITPR3蛋白相对表达量下降(均为P<0.05)。ChIP-seq结果显示,XBP1s能够结合ITPR1的启动子和外显子、ITPR2外显子和ITPR3外显子。结论 XBP1s可能通过直接调控ITPR转录和翻译而影响线粒体相关的内质网膜结构功能,下调XBP1s能够抑制ITPR表达,改善线粒体损伤。

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.

内质网应激头颈部鳞状细胞癌细胞通过外泌体miR⁃26a⁃5p调控PTEN/AKT通路促进巨噬细胞PD⁃L1表达

目的探讨内质网应激(endoplasmic reticulum stress,ERS)的头颈部鳞状细胞癌(head and neck squa⁃mous cell carcinoma,HNSCC)细胞分泌的外泌体miRNA表达变化对巨噬细胞的影响机制。方法本研究已通过单位伦理委员会审查批准。通过蛋白质免疫印迹(Western blot,WB)和实时荧光定量PCR实验(RT⁃qPCR)检测HNSCC肿瘤组织及癌旁组织ERS相关蛋白,包括蛋白激酶R样内质网激酶(protein kinase R⁃like endoplas⁃mic reticulum kinase,PERK)和葡萄糖调节蛋白78(glucose⁃regulated protein 78,GRP78)的表达水平;以500 U/mL干扰素⁃γ(interferon⁃γ,IFN⁃γ)处理人喉鳞癌细胞系HN4细胞48 h,诱发HN4细胞产生内质网应激反应,收集HN4细胞分泌的外泌体,通过生物信息学分析鉴定外泌体的miRNA种类,预测miRNA的靶基因,将巨噬细胞转染miRNA、与收集的外泌体共培养、并敲低巨噬细胞PTEN表达,以WB和RT⁃qPCR检测外泌体miRNA调控的下游信号通路蛋白酪氨酸磷酸酶(phosphate and tension homology deleted on chromsome ten,PTEN)/蛋白激酶B(protein kinase B,AKT)及程序性死亡受体⁃1配体(programmed death receptor ligand 1,PD⁃L1)表达变化。结果HNSCC肿瘤组织相比癌旁组织ERS相关蛋白表达水平升高(P<0.05);RNA测序及实验验证表明ERS的HN4细胞分泌的外泌体miR⁃26a⁃5p表达上调(P<0.05);PTEN是miR⁃26a⁃5p的靶基因,miR⁃26a⁃5p升高巨噬细胞PD⁃L1表达水平,并下调PTEN表达(P<0.05);巨噬细胞与ERS外泌体共培养,miR⁃26a⁃5p和PD⁃L1表达上升,PTEN表达下降,p⁃AKT表达升高(P<0.05);敲低巨噬细胞PTEN表达,PD⁃L1表达上升(P<0.01)。结论ERS的HNSCC细胞通过外泌体miR⁃26a⁃5p调控PTEN/AKT通路及PD⁃L1的表达。

内质网应激IRE1α/XBP-1通路在多巴胺能神经元变性死亡中的作用

目的探讨内质网应激1型跨膜蛋白激酶α(IRE1α)/X盒结合蛋白1(XBP-1)通路在多巴胺能神经元变性死亡中的作用。方法利用内质网应激诱导剂Thapsigargin观察小鼠原代成纤维细胞中IRE1α/XBP-1通路活性。利用IRE1α抑制剂KIRA8探究IRE1α/XBP-1通路在原代中脑多巴胺能神经元变性死亡中作用。结果相比于未处理组细胞,Thapsigargin处理后原代成纤维细胞中内质网应激IRE1α/XBP-1通路明显上调。Thapsigargin处理可显著降低原代中脑多巴胺能神经元的存活率,而KIRA8处理可显著改善Thapsigargin导致的多巴胺能神经元存活率降低。结论内质网应激通过IRE1α/XBP-1通路介导多巴胺能神经元的变性死亡。

Molybdenum and cadmium co-induce necroptosis through Th1/Th2 imbalance-mediated endoplasmic reticulum stress in duck ovaries

Cadmium(Cd)and excess molybdenum(Mo)pose serious threats to animal health.Our previous study has determined that Cd and/or Mo exposure can cause ovarian damage of ducks,while the specific mechanism is still obscure.To further investigate the toxic mechanism of Cd and Mo co-exposure in the ovary,forty 8-day-old female ducks were randomly allocated into four groups for 16 weeks,and the doses of Cd and Mo in basic diet per kg were as follows:control group,Mo group(100 mg Mo),Cd group(4 mg Cd),and Mo+Cd group(100 mg Mo+4 mg Cd).Cadmium sulfate 8/3-hydrate(CdSO_(4)·8/3H_(2)O)and hexaammonium molybdate((NH_(4))_(6)Mo_(7)O_(24)·4H_(2)O)were the origins of Cd and Mo,respectively.At the 16th week of the experiment,all ovary tissues were collected for the detection of related indexes.The data indicated that Mo and/or Cd induced trace element disorders and Th1/Th2 balance to divert toward Th1 in the ovary,which activated endoplasmic reticulum(ER)stress and then provoked necroptosis through triggering RIPK1/RIPK3/MLKL signaling pathway,and eventually caused ovarian pathological injuries and necroptosis characteristics.The alterations of above indicators were most apparent in the joint group.Above all,this research illustrates that Mo and/or Cd exposure can initiate necroptosis through Th1/Th2 imbalance-modulated ER stress in duck ovaries,and Mo and Cd combined exposure aggravates ovarian injuries.This research explores the molecular mechanism of necroptosis caused by Mo and/or Cd,which reveals that ER stress attenuation may be a therapeutic target to alleviate necroptosis.