内质网应激-自噬通路在肝癌细胞凋亡中的作用研究进展

肝癌在我国的发病率和死亡率一直居高不下,其恶性程度较高,早期发现困难且易转移,使得其自然生存期较短。目前对肝癌的治疗仍以手术治疗为主,但术后患者的生存质量并不高,且五年生存率只有20%左右。肝癌的早预防、早发现、早治疗已成为众多研究者亟待解决的问题。研究发现,内质网应激-自噬在肝癌的发生发展中发挥着重要的作用。综合国内外研究,就内质网应激-自噬在肝癌细胞凋亡中的作用作一综述,为肝癌的治疗提供理论支持。     

异硫氰酸苯乙酯通过内质网应激途径促进乳腺癌细胞凋亡及增殖的研究

目的探索异硫氰酸苯乙酯(PEITC)对乳腺癌SK-BR-3细胞凋亡及增殖的影响。方法分别采用不同浓度(0、10、30、50μmol/L)的PEITC处理SK-BR-3细胞,然后采用MTT法和BrdU染色法检测各组细胞的增殖能力,TUNEL法和流式细胞技术检测细胞凋亡,Western blot法和实时荧光定量PCR(qRT-PCR)法检测凋亡相关指标如Bcl-2、Bax、髓细胞白血病基因1(MCL-1)和内质网应激(ERS)相关指标如蛋白激酶R样内质网激酶(PERK)、CCAAT/增强子结合蛋白同源蛋白(CHOP)、肌醇依赖内质网调节细胞核信号激酶1α(IRE1α)、活化转录因子6α(ATF6α)、真核生物翻译起始因子2α亚基(eIF2α)的表达情况。结果与对照组(0μmol/L PEITC处理组)细胞比较,MTT法和BrdU染色法检测结果均显示10、30、50μmol/L PEITC处理组SK-BR-3细胞增殖能力降低且随浓度升高呈依次降低的趋势;TUNEL法和流式细胞技术检测结果均显示10、30、50μmol/L PEITC处理组SK-BR-3细胞的凋亡率升高且随浓度升高呈依次升高的趋势;Western blot和qRT-PCR法检测结果显示抗凋亡指标(Bcl-2、MCL-1)的蛋白和mRNA表达水平均降低且随浓度升高呈依次降低的趋势,而促凋亡指标Bax和ERS相关指标(PERK、CHOP、IRE1α、ATF6α、eIF2α)的蛋白和mRNA表达水平均升高且随浓度升高呈依次升高的趋势。结论从本研究初步研究结果看,PEITC能促进乳腺癌SK-BR-3细胞凋亡、抑制细胞增殖,其可能通过调控凋亡相关指标和ERS相关指标表达来实现。     

内质网应激介导的细胞凋亡与急性肺损伤的研究进展

内质网应激(ERS)介导的细胞凋亡是继死亡受体活化、线粒体损伤途径后新发现的一条重要的细胞凋亡途径,参与多种疾病的发病机制.越来越多的研究表明内质网应激介导的细胞凋亡参与急性肺损伤的发病过程.     

衣霉素诱导内质网应激对树突状细胞成熟分化的影响

目的:内质网应激(ERS)是真核细胞中普遍存在的适应性应激反应,本研究应用ERS特异性诱导剂衣霉素(TM)体外刺激小鼠脾脏树突状细胞(DC),观察DC功能状态的改变情况.方法:分离正常BALB/c小鼠脾脏DC进行体外培养,给予ERS特异性诱导剂TM刺激,观察不同剂量、不同作用时间TM刺激与DC表面共刺激分子表达及分泌功...     

缺血预处理调节内质网应激及促进自噬机制的研究进展

缺血预处理(IPC)对于缺血再灌注损伤(IRI)发生后更迅速的恢复生理功能和组织结构和减少损伤具有重要的作用。细胞受到外界刺激后,内质网应激(ERS)启动,可以通过减少未折叠蛋白的累积并增加未折叠蛋白的降解,保护内质网稳态,泛素化降解系统也参与其中,但当细胞受到外界刺激持续或者过强时,ERS和泛素化降解系统无法消除未折...     

乙型肝炎病毒包膜蛋白诱导肝癌细胞系内质网应激的研究

目的探讨乙型肝炎病毒前-S缺失和野生型外膜大蛋白（LHBs）诱导内质网应激反应。 方法运用脂质体转染技术将pcDNA3.1-S、pcDNA3.1-L、pcDNA3.1-L△30和pcDNA3.1-L△182等真核表达质粒转染至Huh7细胞,并应用qRT-PCR和Western blot方法检测转染细胞前-S1、前-S2、HBsAg和GRP78的mRNA和蛋白表达水平。 结果转染HBV包膜蛋白各组的HBsAg蛋白相对表达量（分别为0.92、0.83、0.91、1.75、2.53和2.06）高于空白对照（0.00）和pcDNA3.1（+）转染组（0.00）（F = 247.38、P = 0.000）;转染HBV包膜蛋白各组的GRP78的蛋白相对表达量（分别为1.4、1.47、1.55、1.75、1.8和1.9）高于空白对照（1.18）和pcDNA3.1(+)转染组（1.23）（F = 11.623、P = 0.000）;且GRP78蛋白相对表达量与前-S1、前-S2抗原和HBsAg表达量呈正相关（相关系数r分别为0.884、0.728和0.816）。 结论前-S缺失型/野生型LHBs及HBsAg均能诱导Huh7细胞发生内质网应激反应,提示其可能参与肝癌发生。     

调节内质网应激对糖尿病小鼠肾脏SET7/9表达的影响

目的 探讨调节内质网应激对小鼠肾组织组蛋白甲基转移酶(HMT) SET7/9表达的影响及意义.方法 db/db小鼠按随机数字表法分为糖尿病肾病(DN)组和甜菜碱治疗(DN+B)组;db/m小鼠作为正常对照(NC)组,每组各18只.实验第4、8、12周末分别采用实时定量PCR和(或)Western印迹法测定小鼠肾组织SET7/9、葡萄糖调节蛋白(GRP)78、H3K4me2和单核细胞趋化蛋白1(MCP-1)表达水平;ELISA法测定24 h尿蛋白排泄率(UPER)和尿MCP-1浓度;全自动生化分析仪检测血糖(BG)、血肌酐、血尿素氮的动态改变;PAS染色观察肾脏病理改变.结果 与NC组比较,DN组BG、BUN、UPER、MCP-1均显著升高(均P＜0.05),且呈时间依赖性.DN组小鼠第4周末开始出现肾小球基底膜增厚、系膜细胞增生改变,第12周末出现明显系膜基质积聚.与NC组比较,DN组肾组织GRP78、SET7/9的mRNA和蛋白质表达水平均显著升高,H3K4me2蛋白水平也显著升高,且呈时间依赖效应.与DN组比较,甜菜碱治疗组小鼠肾小球病变明显减轻,GRP78、SET7/9的mRNA及蛋白表达水平显著降低,BG、BUN、UPER、MCP-1、H3K4me2水平显著降低(均P＜0.05).结论 内质网应激可能是介导糖尿病小鼠肾脏SET7/9表达的上游机制.     

氢气对PC12细胞缺氧复氧时内质网应激的影响

目的 探讨氧气对PC1 2细胞缺氧复氧时内质网应激的影响.方法 PC12细胞采用随机数字表法,将其随机分为4组,正常对照组:细胞常规培养25 h;阳性对照组:细胞正常培养1h后,用氧气饱和的RPM1-1640培养基继续培养24 h;缺氧复氧组:细胞缺氧1h后复氧24 h;氢气组:细胞缺氧1 h后,州氧气饱和的RPM1,1640培氧基复氧24 h.PC12细胞加入含Na2S2O4终浓度为5.0mmol/L的RPMI-1640培养液,5％ CO2培养箱37 ℃孵育1h;更换正常RPMI-1640培养液,继续培养24h,制备PC12细胞缺氧复氧模型.采用WST-1法测定细胞相对增殖率,采用硫代巴比妥酸法测定MDA浓度,采用免疫组化法检测caspase-3表达,采用RT-PCR法检测活化转录因子4(ATF4)mRNA和C/EBP同源蛋白(CHOP)mRNA的表达.结果 与正常对照组和阳性对照组比较,缺氧复氧组细胞相对增殖率降低,MDA浓度升高,caspase-3、ATF4 mRNA和CHOP mRNA的表达七调,氧气组ATF4 mRNA和CHOP mRNA的表达上调(P＜0.05);正常对照组和阳性对照组间细胞相对增殖率、MDA浓度、caspase-3 、ATF4nRNA和CHOP mRNA的表达比较差异无统计学意义(P＞0.05);与缺氧复氧组比较,氢气组细胞相对增殖率升高,MDA浓度降低,caspase-3、ATF4 mRNA和CHOP mRNA的表达下调(P＜0.05).结论 氧气可能通过抑制内质网应激,降低细胞凋亡,减轻PC12细胞缺氧复氧损伤.     

Renal cancer stem cell-derived sEVs impair renal function by inducing renal cell ERS and apoptosis in mice

BackgroundCancer stem cells (CSCs) have been confirmed to participate in tumorigenesis, development, and metastasis, and to affect the local environment in normal tissues. Extracellular vesicles derived from CSCs (CSC-EVs) affect the local environment, contributing to tumor metastasis. However, the effect of small extracellular vesicles (sEVs) from renal CSCs (RCSCs) on renal function has not been studied. This study aimed to establish the impact of RCSC-sEVs on the renal function.MethodsRCSC-sEVs were isolated from cell lines and locally injected into C57 mouse kidneys to observe the effect of RCSC-sEVs on the renal function. 24-hour urinary protein and serum creatinine were examined for renal function evaluation. Periodic Acid-Schiff (PAS) and immunochemistry (IHC) staining were applied for investigations of the pathological changes. Western blot (WB), flow cytometry (FCM), real-time quantitative polymerase chain reaction (RT-qPCR), and TUNEL were employed to assess cell apoptosis and endoplasmic reticulum stress (ERS).ResultsWe found that RCSC-sEVs induced apoptosis and ERS in the mouse kidneys and eventually led to a decrease in the renal function. In vivo, RCSC-sEVs, applied by local injection, induced a continual increase in the 24-hour urinary protein and serum creatinine. In vitro, RCSC-sEVs induced HK2 cell ERS and apoptosis, which was caused by miR-142-3p and was confirmed by antagomir treatment. Further research showed that the miR-142-3p carried by RCSC-sEVs regulated ERp44, thus activating the PERK-CHOP pathway, which induced ERS and led to cell apoptosis.ConclusionsRenal function impairment during tumor development is induced not only by tumor invasion but also by RCSC-sEVs-induced renal cell apoptosis. As a natural vector of miR-142-3p, RCSC-sEVs return to the kidney cells and interfered with the expression of ERp44, inducing ERS and ultimately leading to apoptosis of normal renal cells and renal function impairment.     

Steroidogenesis decline accompanied with reduced antioxidation and endoplasmic reticulum stress in mice testes during ageing

To gain an understanding of the mechanisms by which Leydig cell steroidogenic function degenerates with ageing, we explored steroidogenic gene expression in relation to antioxidation status and endoplasmic reticulum (ER) stress during the ageing of mice. Expression of StAR, P450scc and other steroidogenic enzymes decreased starting at middle age (12‐month‐old) compared to that of the young control (3‐month‐old) mice. The immunohistochemical staining intensity of 3β‐HSD for Leydig cells was significantly weaker in the aged (24‐month‐old) group than that in the young control group. The number of Leydig cells showed no significant difference between the groups. A progressive reduction in antioxidants MnSOD and GPx4 was observed in the testicular tissue with down‐regulated SIRT1 protein level in the middle‐aged and aged (24‐month‐old) mice. The number of testicular macrophages was significantly higher in the aged group than that in the middle‐aged and young mice. Age‐associated up‐regulation of ER stress markers such as GRP78 and Chop was observed. These results suggested that oxidative stress and ER stress might play a role in the deficit of Leydig cell steroidogenic function during ageing.     

胃饥饿素对衣霉素诱导成骨细胞内质网应激的影响

目的建立衣霉素诱导成骨细胞内质网应激的模型,探讨胃饥饿素(ghrelin)对衣霉素诱导成骨细胞内质网应激的影响。方法选取小鼠成骨细胞MC3T3-E1为研究对象,(1)分别将不同浓度的衣霉素(0、0. 5、1、1. 5μg/mL)加入成骨细胞培养基中,分别孵育24 h后,采用CCK-8法检测细胞增殖活性;二氯二氢荧光素-乙酰乙酸酯探针(DCFH-DA)检测胞内活性氧(ROS)的含量; real-time quantitative PCR(qRT-PCR)检测各组细胞内内质网应激相关标志性基因BIP、CHOP、caspase-12 mRNA的表达。最后选择衣霉素作用最敏感浓度1. 5μg/mL,建立成骨细胞内质网应激的模型。(2)观察ghrelin对成骨细胞内质网应激的影响。分别用不同浓度的ghrelin(0、10~(-11)、10~(-9)、10~(-7)mmol/L)预处理成骨细胞4 h后,加入1. 5μg/mL的衣霉素诱导成骨细胞内质网应激。培养结束后,利用上述方法检测细胞增殖活性、胞内ROS的含量、内质网应激相关标志性基因的表达。结果与对照组相比,不同浓度的衣霉素干预成骨细胞24 h后,细胞增殖和存活率呈浓度依赖性明显降低。1. 0μg/mL和1. 5μg/mL的衣霉素干预细胞24 h后细胞增殖和存活率的降低有统计学意义(P0. 05),而0. 5μg/mL的衣霉素干预后无统计学意义(P0. 05);与对照组相比,胞内ROS的含量随浓度的增加逐渐增加(P0. 05); qRT-PCR结果显示,与对照组相比,不同浓度衣霉素干预细胞后CHOP mRNA的表达均有明显的提高且均有统计学意义(P0. 05),而BIP、caspase-12 mRNA的表达只在1. 0μg/mL和1. 5μg/mL衣霉素干预后有统计学意义(P0. 05),0. 5μg/mL衣霉素干预后无统计学意义(P0. 05);(2)与单纯1. 5μg/mL衣霉素相比,用不同浓度的ghrelin预处理成骨细胞4 h后再加1. 5μg/mL衣霉素,发现细胞增殖和存活率随ghrelin浓度增高而增加,10~(-9)、10~(-7)mmol/L ghrelin预处理后有统计学意义(P0. 05),而10~(-11)mmol/L ghrelin预处理后无统计学意义(P0. 05);与单纯1. 5μg/mL衣霉素相比,不同浓度的ghrelin预处理后胞内ROS的含量减少,且均有统计学意义(P0. 05); qRT-PCR结果显示,在10~(-9)和10~(-7)mmol/L ghrelin预处理后,CHOP mRNA表达降低有统计学意义(P0. 05),而10~(-11)mmol/L的ghrelin预处理后无统计学意义(P0. 05)。对于BIP和caspase-12 mRNA的表达,不同浓度的ghrelin预处理后均有统计学意义(P0. 05)。结论衣霉素可以诱导成骨细胞内质网应激的发生,ghrelin在一定程度上可以抑制成骨细胞内质网应激。     

N-acetylcysteine protects against cadmium-induced germ cell apoptosis by inhibiting endoplasmic reticulum stress in testes

Cadmium (Cd) is a reproductive toxicant that induces germ cell apoptosis in the testes. Previous studies have demonstrated that endoplasmic reticulum (ER) stress is involved in Cd-induced germ cell apoptosis. The aim of the present study was to investigate the effects of N-acetylcysteine (NAC), an antioxidant, on Cd-induced ER stress and germ cell apoptosis in the testes. Male CD-1 mice were intraperitoneally injected with CdCl₂ (2.0 mg kg⁻¹). As expected, acute Cd exposure induced germ cell apoptosis in the testes, as determined by terminal dUTP nick-end labelling (TUNEL). However, the administration of NAC alleviated Cd-induced germ cell apoptosis in the testes. Further analysis showed that NAC attenuated the Cd-induced upregulation of testicular glucose-regulated protein 78 (GRP78), an important ER molecular chaperone. Moreover, NAC inhibited the Cd-induced phosphorylation of testicular eukaryotic translation initiation factor 2α (eIF2α), a downstream target of the double-stranded RNA-activated kinase-like ER kinase (PERK) pathway. In addition, NAC blocked the Cd-induced activation of testicular X binding protein (XBP)-1, indicating that NAC attenuates the Cd-induced ER stress and the unfolded protein response (UPR). Interestingly, NAC almost completely prevented the Cd-induced elevation of C/EBP homologous protein (CHOP) and phosphorylation of c-Jun N-terminal kinase (JNK), two components of the ER stress-mediated apoptotic pathway. In conclusion, NAC protects against Cd-induced germ cell apoptosis by inhibiting endoplasmic reticulum stress in the testes.     

TSG-6 inhibits hypertrophic scar fibroblast proliferation by regulating IRE1α/TRAF2/NF-κB signalling

TNF-stimulated gene (TSG-6) was reported to suppress hypertrophic scar (HS) formation in a rabbit ear model, and the overexpression of TSG-6 in human HS fibroblasts (HSFs) was found to induce their apoptotic death. The molecular basis for these findings, however, remains to be clarified. HSFs were subjected to TSG-6 treatment. Treatment with TSG-6 significantly suppressed HSF proliferation and induced them to undergo apoptosis. Moreover, TSG-6 exposure led to reductions in collagen I, collagen III, and α-SMA mRNA and protein levels, with a corresponding drop in proliferating cell nuclear antigen (PCNA) expression indicative of impaired proliferative activity. Endoplasmic reticulum (ER) stress was also suppressed in these HSFs as demonstrated by decreases in Bip and p-IRE1α expression, downstream inositol requiring enzyme 1 alpha (IRE1α) -Tumor necrosis factor receptor associated factor 2 (TRAF2) pathway signalling was inhibited and treated cells failed to induce NF-κB, TNF-α, IL-1β, and IL-6 expression. Overall, ER stress was found to trigger inflammatory activity in HSFs via the IRE1α-TRAF2 axis, as confirmed with the specific inhibitor of IRE1α STF083010. Additionally, the effects of TSG-6 on apoptosis, collagen I, collagen III, α-SMA, and PCNA of HSFs were reversed by the IRE1α activator thapsigargin (TG). These data suggest that TSG-6 administration can effectively suppress the proliferation of HSFs in part via the inhibition of IRE1α-mediated ER stress-induced inflammation (IRE1α/TRAF2/NF-κB signalling).     

Immunohistochemical study of phospholipase A2 in boyine prostate

Approximately 1,100-fold purified phospholipase A₂ (PLA₂) from bovine prostate was injected into rabbit to prepare polyclonal antibodies. Antibodies produced showed specific immunoprecipitation only with the purified enzyme, as well as with homogenate of bovine prostatic tissue. By Western blot analysis or by immunodiffusion test, no cross-reactivity with PLA₂ purified from human seminal plasma, bovine pancreas, Crotalus adamanteus venom, or with partially purified PLA₂ from bovine seminal vesicle fluid or Cowper's glands was observed. Using the indirect peroxidase technique, PLA₂ was localized in the cytoplasm of bovine prostatic epithelial cells. By immunogold microscopy, this enzyme was directly visualized inside the lysosomes, as well as in the endoplasmic reticulum of the glandular epithelial cells. Enzyme activity was localized in two principal subcellular sites: the mitochondria and lysosome-enriched fraction, and in the microsomal fraction. © 1993 Wiley-Liss, Inc.