葡萄糖调节蛋白78与肿瘤内质网关系研究进展

GRP78存在于人体内质网中，并参与蛋白质成熟、装配、折叠等重要生物学过程。当细胞受到内部或外界刺激时，内质网腔中未折叠和错误折叠的蛋白质会大量聚集造成内质网应激，细胞则通过未折叠蛋白应答(UPR)来缓解这种应激状态，以恢复内质网稳定。这种情况同样会发生在肿瘤细胞中，在肿瘤细胞的内质网处于应激状态时，会伴有过度增殖、缺血、缺氧、代谢异常等一系列反应，此时GRP78会被激活来缓解内质网腔内压力。但实际上过多的GRP78会带来抑制凋亡、产生耐药性等负面作用。本文主要对GRP78与肿瘤内质网之间的关系展开综述。     

三七总皂苷抑制毒胡萝卜素诱导Bip/GRP78和caspase-12的表达

目的探索三七总皂苷在内质网(ER)应激条件下的细胞保护作用,分析三七总皂苷的抗肝细胞凋亡作用的机制。方法毒胡萝卜素(TG)诱导人类肝脏来源的细胞株Huh7细胞形成内质网应激介导的细胞凋亡。设置空白对照,三七总皂苷,毒胡萝卜素,及三七总皂苷加毒胡萝卜素不同处理细胞,加入细胞培养TG浓度5μmol/L,三七总皂苷浓度150μmol/L,培养24 h。荧光显微镜观察细胞形态学形态学,流式细胞仪测定细胞凋亡,免疫印记法检测Bip/GRP78和pro caspase-12,细胞caspase-3/7活性分析。结果毒胡萝卜素处理细胞染色质固缩,向外周聚集,周边化。形成很多颗粒物质。大量细胞核破裂形成碎片,核解体。三七总皂苷共同处理的细胞改善了细胞器的完整性,减少形态学改变,DAPI染色颗粒物质减少了45%。流式细胞仪测定显示三七总皂苷处理减少了2.5倍毒胡萝卜素引起的细胞凋亡。三七总皂苷减少TG诱导内质网应激标志物Bip/GRP78产生,降低pro caspase-12向caspase-12转化。减少caspase-3/7活性50%。结论三七总皂苷具有细胞保护作用,对抗内质网应激介导的细胞凋亡。三七总皂苷的抗凋亡作用的机制与稳定内质网内环境,抵消TG-诱导的内质网应激Bip/GRP78产生,降低pro caspase-12向caspase-12转化有关。这些研究结果有助于我们了解三七总皂苷对内质网应激相关的肝脏疾病的治疗作用机制。     

胱硫醚-β-合酶/硫化氢系统在鱼藤酮诱导PC12细胞损伤中的变化

目的动态研究胱硫醚-β-合酶/硫化氢(CBS/H2S)系统在鱼藤酮诱导PC12细胞损伤中的变化。方法鱼藤酮诱导具有多巴胺能神经元特性的PC12细胞损伤作为帕金森病(Parkinson's disease,PD)细胞模型,免疫印迹法(Western blot)检测CBS的表达;亚甲基蓝分光光度计法检测细胞内CBS酶活性及硫化氢(hydrogen sulfide,H2S)的生成;应用CCK-8比色法检测细胞存活率;GSH检测试剂盒检测细胞内GSH含量。结果鱼藤酮损伤6和12 h组,PC12细胞内CBS的表达和活性升高,H2S生成量增加;而在24和48 h组,CBS表达和活性则明显降低,H2S的生成量明显减少;鱼藤酮可以时间依赖性的降低细胞存活率及减少细胞内GSH的含量。结论鱼藤酮刺激引起内源性CBS的表达和活性先升高后降低,H2S的生成先增加后减少,这可能与PC12细胞对抗鱼藤酮诱导的氧化应激损伤有关。     

天麻素通过抑制细胞凋亡保护谷氨酸诱导的PC12细胞损伤

目的：研究天麻素（Gastrodin）对谷氨酸诱导的大鼠肾上腺嗜铬细胞瘤PC12细胞损伤的影响及可能机制。方法：以谷氨酸建立体外培养PC12细胞损伤模型并采用MTT比色法测定细胞存活率;AO/EB双染法经荧光显微镜观察细胞凋亡形态;采用流式细胞术检测细胞内活性氧含量以及Annexin V/PI染色后的细胞凋亡率;Western blot法检测细胞内Caspase-3蛋白表达。结果：天麻素可明显抑制谷氨酸诱导的PC12细胞凋亡,在0.1～10μmol/L剂量呈一定的量效关系;同时,天麻素可明显抑制谷氨酸引起的活性氧（ROS）的累积,降低谷氨酸诱导的活性Caspase-3蛋白的表达,降低PC12细胞的凋亡率,在0.1～10μmol/L剂量呈量效相关性。结论：在一定剂量范围内,天麻素对谷氨酸损伤的PC12细胞具有保护作用,其机制可能与减少ROS的生成,阻止氧化损伤的发生,抑制Caspase-3途径依赖的细胞凋亡相关。     

铅对大鼠C6细胞GRP78表达的影响

目的观察不用时间点暴露于不同剂量醋酸铅后大鼠神经胶质瘤C6细胞葡萄糖调节蛋白78(glucoseregulated protein 78,GRP78)表达量的影响,探讨不同时间不同剂量铅暴露对内质网应激反应的影响。方法Wistar大鼠神经胶质瘤C6细胞培养于含醋酸铅的培养液中,分别于不同时间终止染铅,用Western印迹法检测内质网GRP78表达量。结果(1)0.2μmol/L染铅组:7和30 d GRP78蛋白表达显著增高,其余各个时间点均无显著性变化;(2)1.0μmol/L染铅组:1 d后GRP78蛋白表达量开始显著增高,染铅30 d时已达到染铅前的6.3倍;(3)2.0μmol/L染铅组:0.5 h起GRP78表达量即显著增高,染铅7 d时达到高峰,是染铅前的4.3倍,到30 d时表达量又下降为染铅前的2.6倍。结论铅可以使Wistar大鼠神经胶质瘤细胞内质网上的GRP78蛋白应激性表达增加,内质网上的GRP78是铅的重要蓄积库。     

热休克蛋白90在硫化氢保护PC12细胞对抗化学性缺氧损伤中的作用

目的探讨热休克蛋白90(Hsp90)在硫化氢(H2S)保护PC12细胞对抗氯化钴(CoCl2)引起的化学性缺氧损伤中的作用。方法在PC12细胞建立H2S预处理对抗CoCl2诱导PC12细胞损伤的实验模型。应用细胞计数试剂盒8(CCK-8)检测细胞存活率;Hoechst 33258染色荧光显微镜照相术检测凋亡PC12细胞的形态学改变;应用碘化丙啶(PI)染色流式细胞术检测细胞凋亡率;免疫印迹法(Western blot)检测Hsp90的表达。结果H2S的供体400μmol·L-1硫氢化钠(NaHS)可上调PC12细胞Hsp90的表达,NaHS作用3h,Hsp90表达达最高峰,作用24h时表达恢复到基础水平;NaHS也能明显地增加CoCl2引起的Hsp90的表达上调。NaHS预处理能对抗CoCl2引起的PC12细胞损伤,提高细胞存活率,降低细胞凋亡率。Hsp90抑制剂17-丙烯胺基-17-去甲氧基格尔德霉素(17-AAG)可拮抗NaHS预处理对Hsp90表达的上调作用,并明显地减弱H2S诱导的适应性细胞保护作用。结论H2S能保护PC12细胞对抗CoCl2诱导的低氧损伤作用,诱导Hsp90表达上调可能是其细胞保护机制之一。     

Daidizein对β淀粉样蛋白诱导的PC12细胞损伤的保护作用

目的研究Daid ize in对β淀粉样蛋白(Aβ_1-42)诱导的PC12细胞损伤的保护作用。方法用Aβ_1-42处理诱导体外培养的PC12细胞损伤,并用不同浓度的Daid ize in进行保护,显微观察细胞形态的改变,LDH法和MTT法检测细胞膜的损伤情况。结果保护组细胞数比损伤组显著增多,受损变圆的细胞数较少,细胞形态明显改善,保护组上清液LDH活性显著低于损伤组(P<0.01),MTT值显著高于损伤组(P<0.01)。结论Daid ize in对Aβ_1-42诱导的PC12细胞损伤具有显著的保护作用。     

辣椒素通过调节GRP78触发内质网应激对人急性髓系白血病HL-60细胞凋亡的影响

目的 探讨辣椒素通过调节GRP78触发内质网应激对人急性髓系白血病HL-60细胞凋亡的影响及机制.方法 CCK-8筛选辣椒素的最佳作用浓度;Wright-Giemsa染色观察HL-60细胞的形态;Western blot检测GRP78蛋白的表达量;Annexin V-FITC/PI双染法检测HL-60细胞的凋亡率.结果...     

Daidizein对β淀粉样蛋白诱导的PC12细胞损伤的保护作用

目的研究Daid ize in对β淀粉样蛋白(Aβ1-42)诱导的PC12细胞损伤的保护作用。方法用Aβ1-42处理诱导体外培养的PC12细胞损伤,并用不同浓度的Daid ize in进行保护,显微观察细胞形态的改变,LDH法和MTT法检测细胞膜的损伤情况。结果保护组细胞数比损伤组显著增多,受损变圆的细胞数较少,细胞形态明显改善,保护组上清液LDH活性显著低于损伤组(P<0.01),MTT值显著高于损伤组(P<0.01)。结论Daid ize in对Aβ1-42诱导的PC12细胞损伤具有显著的保护作用。     

刺五加有效部位干预MPP+诱导PC12细胞损伤中LRRK 2的表达

摘 要 目的：观察刺五加有效部位对MPP+诱导损伤的PC12细胞LRRK 2蛋白表达的影响,探讨其神经保护的作用机制。方法: 采用MPP+作用于PC12细胞构建帕金森病的细胞模型,以刺五加有效部位进行给药干预;采用MTT法测细胞存活率;荧光定量PCR法检测细胞LRRK 2 mRNA的表达水平,Western blot法和免疫细胞化学法检测细胞LRRK 2蛋白的表达。结果: 给药组与模型组比较,细胞存活率明显提高,差异有统计学意义（P＜0.01）;给药组与模型组比较,细胞中LRRK 2的蛋白表达水平明显降低,差异有统计学意义（P＜0.01）。结论:刺五加有效部位对PD细胞模型具有保护作用,刺五加有效部位下调LRRK2 蛋白的表达水平可能是其起到保护作用的机制之一。     

甲醛对PC12细胞内源性H_2S生成的影响

目的观察甲醛对PC12细胞内源性硫化氢(hydrogensulfide,H2S)生成的影响,以探讨甲醛损伤PC12细胞的新机制。方法 RT-PCR方法检测PC12细胞的胱硫醚-β-合酶(cystathionine-beta-synthase,CBS)mRNA的表达;亚甲基蓝分光光度计法检测PC12细胞CBS活性及PC12细胞内源性H2S的含量;MTT法观察PC12细胞的存活率。结果甲醛可以抑制PC12细胞CBS的表达及其活性,减少内源性H2S的生成;甲醛可以明显地降低PC12细胞的存活率。结论甲醛能抑制CBS的表达和活性,减少内源性H2S生成,这可能与其损伤PC12细胞有关。     

2-脱氧葡萄糖对脑缺血再灌注模型大鼠脑组织中葡萄糖调节蛋白78及天冬氨酸半胱氨酸蛋白酶-12的影响研究

目的:探讨2-脱氧葡萄糖(2-DG)对脑缺血再灌注(IR)模型大鼠脑组织中葡萄糖调节蛋白78(GRP78)及天冬氨酸半胱氨酸蛋白酶-12(caspase-12)的影响。方法:取大鼠随机分为假手术组、模型组和2-DG组(2-DG100mg·kg-1),每组60只,后2组建立局灶性脑IR模型,假手术组行手术但不插入线栓。建模前7d开始给药,每天1次,连续7d,考察建模后3、6、12、24、48h各组大鼠海马CA1区细胞凋亡情况(阳性细胞率)、GRP78和caspase-12蛋白及其mRNA的表达情况。结果:与假手术组比较,模型组和2-DG组阳性细胞率、GRP78和caspase-12蛋白及其mRNA表达均明显升高(P均<0.01);与模型组比较,2-DG组阳性细胞率、cas-pase-12蛋白及其mRNA表达明显降低,GRP78蛋白及其mRNA表达明显升高(P<0.05或P<0.01)。结论:2-DG可能通过上调GRP78和下调caspase-12的表达来预防脑IR损伤。     

Propofol attenuates oxidative stress-induced PC12 cell injury via p38 MAP kinase dependent pathway

Aim： To investigate the neuroprotective effect of propofol and its intracellular mechanism on neurons in vitro. Methods： Cell viability was determined with 3-（4, 5-dimethylthiazol-2-yl）-2,5-diphenyl tetrazolium bromide reduction. Apoptotic cell death was determined by Hoechst 33258 staining and a fluorescence-activated cell sorter. The caspase-3 activity was measured by fluorometric assay. Mitogenactivated protein （MAP） kinase phosphorylation was detected with Western blotting. Results： The pretreatment of rat pheochromocytoma cell line PC 12 with propofol （1-10 μmol/L） resulted in a significant recovery from hydrogen peroxide （H2O2）-induced cell death and the inhibition of H2O2 induced caspase-3 activation and PC12 cell apoptosis. Propofol inhibited the H2O2-induced p38 MAP kinase, but not c-Jun N-terminal kinase or extracellular signal-regulated kinase 1 and 2 activations. Conclusion： Propofol might attenuate H2O2-induced PC 12 cell death through the inhibition of signaling pathways mediated by the p38 MAP kinase.     

砷及MPST过表达对SH-SY5Y细胞GRP78/CHOP通路的影响

目的探讨内质网应激是否参与亚砷酸钠(NaAsO2)神经毒性损伤,明确3-巯基丙酮酸硫转移酶(3-mercaptopyruvate sulfurtransferase,MPST)过表达是否调节砷诱导的内质网应激。方法通过构建MPST基因慢病毒表达载体来获得稳定表达外源MPST基因的SH-SY5Y细胞株作为SH-MPST过表达组,另设空载体转染细胞为SH-PEB组,染砷组(NaAsO2组),内质网应激阻断剂TUDCA组,TUDCA预处理染砷组。Western blot法分别检测过表达MPST、染砷及TUDCA预处理后细胞内GRP78和CHOP蛋白表达的变化。结果单纯MPST过表达不影响SH-SY5Y细胞内GRP78、CHOP蛋白的表达水平;经NaAsO2处理后,SH-PEB细胞内GRP78、CHOP蛋白明显上调(P<0.01),而被内质网应激阻断剂TUDCA所拮抗;MPST过表达则抑制砷对GRP78、CHOP蛋白的上调(P<0.01);然而,TUDCA预处理则明显逆转MPST过表达对GRP78、CHOP蛋白的影响(P<0.01)。结论GRP78/CHOP内质网应激通路参与了砷诱导的神经毒性损伤;MPST过表达可降低砷诱导的内质网应激水平。     

d-Limonene protects PC12 cells against corticosterone-induced neurotoxicity by activating the AMPK pathway

The stress-induced hormone corticosterone initiates oxidative stress and inflammatory responses, culminating in cell apoptosis and neurological changes. We assessed the effects of d-Limonene on a PC12 cellular model of corticosterone-induced neurotoxicity, and whether these effects involved the AMP-activated protein kinase (AMPKα) pathway. PC12 cells were treated with corticosterone with or without d-limonene for 24 h. Western blots were performed to measure activation of AMPK pathway members [Silent mating type information regulation 2 homolog-1 (SIRT1), AMPKα, and nuclear factor (NFκB)], reactive oxygen species, inflammatory cytokines, and markers of apoptosis. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) was used to measure cell death after treatment. d-Limonene reversed the effects of corticosterone on PC12 cells: it decreased the levels of malondialdehyde (MDA) and nitric oxide (NO), activities of NADPH oxidase (p67-phox and p47-phox), expression of pro-inflammatory markers [inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin 6 (IL-6), interleukin 1β (IL-1β), and tumor necrosis factor α (TNF-α)], and expression of pro-apoptotic proteins [Bcl2 associated with X protein (Bax) and cleaved caspase-3)]. d-Limonene also increased levels of the antioxidant enzymes superoxide dismutase 1 (SOD1) and heme oxygenase 1 (HO-1) and the anti-apoptotic protein Bcl-2 while decreasing the number of TUNEL-positive cells. d-limonene significantly activated AMPKα and suppressed NF-κB nuclear translocation through up-regulation of SIRT1. Addition of compound C, an AMPK inhibitor, severely weakened these neuroprotective effects of d-limonene. d-Limonene has a neuroprotective effect on corticosterone-induced PC12 cell injury induced by activating the AMPKα signaling pathway, and thereby inhibiting reactive oxygen species and inflammatory factors. These data suggest that d-limonene might protect against neuronal death to improve depressive symptoms.     

醋酸锰对PC12细胞损伤作用的研究

目的研究醋酸锰对PC12细胞的损伤作用机制。方法不同浓度醋酸锰处理PC12细胞24h后,MTT法检测细胞存活率,Hoechst 33258和DNA凝胶电泳的方法检测细胞凋亡,利用高效液相色谱—电化学方法(HPLC-ECD)检测细胞内儿茶酚异喹啉物质的生成,同时测定细胞内丙二醛(MDA)和羟自由基的生成量。结果不同水平的醋酸锰处理使PC12细胞存活率下降且呈浓度依赖性(P<0.05)。Hoechst 33258和DNA凝胶电泳结果表明细胞发生了凋亡。MDA和羟自由基的含量与对照相比明显升高(P<0.05),多巴胺(DA)的含量呈下降趋势(P<0.05)。Sal(6,7-二羟基-1,2,3,4-四氢异喹啉,Sal)和NMSal(N-甲基-6,7-二羟基-1,2,3,4-四氢异喹啉,NMSal)的含量则随锰浓度的增加而增加(P<0.05)。结论过量的醋酸锰导致PC12细胞产生高氧化应激水平,从而生成了一系列儿茶酚异喹啉物质,对细胞产生损伤作用,这可能是三价锰发挥损伤作用的途径之一。     

GRP78在二型糖尿病(T2DM)及其并发症中表达研究进展

糖尿病是一种常见的慢性疾病,有许多并发症,涉及多个器官和组织。葡萄糖调节蛋白78(GRP78)是内质网应激最敏感的伴侣蛋白。糖尿病及其并发症与内质网应激有着密切联系。研究GRP78表达特征及机制,对糖尿病及其并发症的发病机制及治疗有重要意义。文章就GRP78在二型糖尿病(T2DM)及其并发症中的表达和作用进行了综述。     

Neuroprotective effects of pramipexole against tunicamycin‐induced cell death in PC12 cells

• 1 Pramipexole (PPX), a dopamine D2 and D3 receptor agonist, exerts neuroprotective effects via both dopamine receptor‐mediated and non‐dopaminergic mechanisms. In the present study, we demonstrate that PPX reduces the toxicity of tunicamycin, a typical endoplasmic reticulum (ER) stressor, in PC12h cells, a subline of PC12 cells.
• 2 The PC12h cells were treated with 300 μmol / L PPX in the presence of 0.5 μmol / L tunicamycin for 24 h. The neuroprotective effects of PPX against tunicamycin‐induced cell death were evaluated using 3‐(4,5‐dimethyl‐2 thiazoyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) release assays, Hoechst 33258 staining and western blot analysis.
• 3 Tunicamycin (0.2, 0.3 and 0.5 μg / mL) dose‐dependently decreased MTT activity and increased LDH release from PC12h cells. Treatment with 300 μmol / L PPX rescued the tunicamycin‐induced decrease in cell viability.
• 4 Spiperone (10 μmol / L), a dopamine D2 and D4 receptor antagonist, had no effect on PPX neuroprotection against tunicamycin in these cells. Marker proteins of ER stress and apoptosis are known to be upregulated by tunicamycin, but we detected no significant effects of PPX on these factors.
• 5 In conclusion, we speculate that a combination of several mechanisms may be involved in PPX‐induced neuroprotection.

     

Parkin represses 6-hydroxydopamine-induced apoptosis via stabilizing scaffold protein p62 in PC12 cells

Aim:

Parkin has been shown to exert protective effects against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in different models of Parkinson disease. In the present study we investigated the molecular mechanisms underlying the neuroprotective action of parkin in vitro.

Methods:

HEK293, HeLa and PC12 cells were transfected with parkin, parkin mutants, p62 or si-p62. Protein expression and ubiquitination were assessed using immunoblot analysis. Immunoprecipitation assay was performed to identify the interaction between parkin and scaffold protein p62. PC12 and SH-SY5Y cells were treated with 6-OHDA (200 μmol/L), and cell apoptosis was detected using PI and Hoechst staining.

Results:

In HEK293 cells co-transfected with parkin and p62, parkin was co-immunoprecipitated with p62, and parkin overexpression increased p62 protein levels. In parkin-deficient HeLa cells, transfection with wild-type pakin, but not with ligase activity-deficient pakin mutants, significantly increased p62 levels, suggesting that parkin stabilized p62 through its E3 ligase activity. Transfection with parkin or p62 significantly repressed ERK1/2 phosphorylation in HeLa cells, but transfection with parkin did not repress ERK1/2 phosphorylation in p62-knockdown HeLa cells, suggesting that p62 was involved in parkin-induced inhibition on ERK1/2 phosphorylation. Overexpression of parkin or p62 significantly repressed 6-OHDA-induced ERK1/2 phosphorylation in PC12 cells, and parkin overexpression inhibited 6-OHDA-induced apoptosis in PC12 and SH-SY5Y cells.

Conclusion:

Parkin protects PC12 cells against 6-OHDA-induced apoptosis via ubiquitinating and stabilizing scaffold protein p62, and repressing ERK1/2 activation.