IRE1介导的内质网应激在实验性暴发性肝衰竭肝细胞凋亡中的作用及其机制
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摘要
目的:暴发性肝衰竭(fulminant hepatic failure,FHF)是由多种原因引起的大量肝细胞急性坏死和严重肝功能损伤所致的临床综合征。FHF具有起病急、进展迅速、并发症多、病死率高、预后极差的特点,是当前国内外研究的热点和难点,其发病机制仍未完全阐明。
目前研究认为,FHF与肝细胞凋亡密切相关,但其具体作用机制尚不清楚。内质网应激是除死亡受体活化和线粒体损伤外又一条新的介导细胞凋亡的信号传导通路,与许多肝脏疾病相关,是目前国内外研究的热点。
内质网(endoplasmic reticulum,ER)是细胞内最大的膜网络结构,具有合成、加工蛋白参与代谢和细胞信号处理功能。内质网应激(endoplasmic reticulum stress,ERS)是指由于某种原因导致细胞内质网生理功能发生紊乱,钙稳态失衡,错误折叠或未折叠的蛋白质在内质网腔内聚集的一种亚细胞器的病理状态,是使错误的蛋白质恢复正确构象并能够进一步加工所必需的步骤,是一种细胞自我保护的机制。但是过强或长时间的ERS则可能导致细胞凋亡。内质网应激反应过程中,当大量膜蛋白在内质网沉积时,可激活内质网超负荷反应(ER overload response,EOR),NF-κB(nuclear factorκB)被活化,在EOR信号通路中需肌醇蛋白1(inositol requiring 1,IRE1)处于中心地位,IRE1是内质网跨膜蛋白,参与ERS的适应、警戒、凋亡三个阶段,ERS时IRE1活化,聚集肿瘤坏死因子受体相关因子2(tumor necrosis factor receptor associated factor 2, TRAF2),引起NF-κB活化。NF-κB是一种广泛存在于细胞中具有多向性调节作用的核转录因子,与细胞凋亡关系密切,其参与多种凋亡相关基因的转录调控,具有抑制细胞凋亡和促进细胞凋亡的双重作用。而且有研究表明TRAF2可通过激活NF-κB诱导激酶(NF-κB inducing kinase, NIK)激活NF-κB。Caspase-12是内质网膜结合蛋白,能够且仅被ERS介导的凋亡活化。近年来发现ERS介导的细胞凋亡与乙型肝炎、非酒精性脂肪性肝病、阿尔茨海默病和糖尿病等密切相关,而在暴发性肝衰竭肝细胞凋亡中的作用如何报道较少。本研究应用D-氨基半乳糖( D-galactosamine,D-GalN )和脂多糖(lipopolysaccharide,LPS)制造大鼠暴发性肝衰竭模型,对照组同步注射同样体积的生理盐水进行比较。通过采用免疫组化及反转录多聚酶链反应(reverse transcriptase polymerase chain reaction,RT-PCR)方法检测两组肝组织Caspase-12、IRE1、NF-κB、NIK表达情况,采用常规苏木素-伊红(hematoxylin-eosin,HE)染色,流式细胞学技术(flow cytometry,FCM)观察肝细胞凋亡情况,分析内质网跨膜蛋白IRE1与肝细胞凋亡程度、Caspase-12的相互关系及Caspase-12与肝细胞凋亡程度的相互关系,探讨内质网应激在暴发性肝衰竭肝细胞凋亡中的作用及机制;分析NF-κB活性与肝细胞凋亡程度、IRE1及NIK的关系,探讨IRE1介导NF-κB激活的信号转导通路在暴发性肝衰竭发病机制中的作用,为阐明暴发性肝衰竭的发病机制和防治提供理论依据。
方法: 1研究对象:雄性Wistar大鼠60只,清洁级,体重180-200g。大鼠禁食24小时后,随机分为2组,即模型组和对照组,每组30只。模型组腹腔注射D-GalN800mg/kg,之后皮内注射LPS10ug/kg制造暴发性肝衰竭模型,对照组注射同样体积的生理盐水。2标本采集和保存:从对照组、模型组各取6只分别于给D-GalN +LPS后2、4、8、12、24小时行股静脉放血处死,立即剖腹取出肝脏,取适量肝组织分别固定于10%中性甲醛液、70%乙醇中,部分肝组织经液氮速冻后存放于-80℃冰箱。3 HE染色光镜下观察肝组织病理学变化。4采用免疫组织化学pv法检测肝组织中Caspase-12、IRE1、NF-κB p65、NIK蛋白表达情况。5采用RT-PCR方法检测肝组织中Caspase-12、IRE1、NF-κB p65、NIKmRNA的表达情况。6应用流式细胞学技术测定肝细胞凋亡率(apoptotic rate,AR)。7统计学处理:应用spss13.0统计软件进行统计学分析,采用方差分析,秩和检验和Spearman秩相关分析对数据进行统计。
结果:
1一般状况观察:模型组大鼠随着给药时间的延长出现活动减少、反应迟钝、毛发竖立、嗜睡、昏迷等情况。对照组大鼠一般状况较好。
2肝组织形态学观察:(1)大体标本观察:模型组大鼠肝脏随着给药时间的延长出现充血、出血点及淤血、淤斑逐渐增多,到24小时肝组织呈暗红色,大片出血,淤血严重。对照组大鼠肝脏基本正常。(2)HE染色:模型组大鼠肝组织病理显示给药后2小时肝小叶完整,肝索排列规则;4小时可见肝细胞嗜酸性变及凋亡小体,少量炎细胞浸润;8小时可见炎细胞浸润及多量凋亡小体;12小时肝细胞片状坏死,可见炎细胞浸润,多量凋亡肝细胞;24小时肝组织大片坏死,出血严重,残留散在肝细胞及凋亡小体。对照组大鼠肝组织基本正常。
3细胞凋亡的变化:流式细胞学技术检测细胞凋亡率结果显示在模型组肝细胞凋亡率随着给药时间延长呈现上升趋势(P <0.05);对照组肝细胞凋亡率较低(P>0.05),在4h、8h、12h、24h较模型组细胞凋亡率减少(P <0.05)。
4肝组织Caspase-12检测结果:(1)免疫组化方法:模型组Caspase-12蛋白主要表达在肝细胞浆,随着给药时间的延长表达增多(P<0.01);对照组肝细胞胞浆Caspase-12蛋白未见明显表达。(2)RT-PCR结果显示:模型组Caspase-12 mRNA表达随着给药时间的延长而增加,8h达高峰,以后逐渐下降,各时间点差别有统计学意义(P<0.01),对照组Caspase-12 mRNA各个时间点弱表达(P>0.05),在4h、8h、12h、24h,模型组Caspase-12 mRNA表达均明显高于对照组(P<0.05)。
5肝组织IRE1α检测结果:(1)免疫组化方法:模型组IRE1α蛋白主要在肝细胞浆表达,随着给药时间的延长,IRE1α蛋白表达明显增多(P<0.01);对照组肝细胞胞质IRE1α蛋白未见明显表达。(2)RT-PCR结果显示:模型组随着给药时间的延长IRE1αmRNA表达增高(P<0.01);对照组IRE1αmRNA各个时间点弱表达(P>0.05),各时间点模型组IRE1αmRNA表达均明显高于对照组(P<0.05)。
6肝组织NF-κBp65检测结果:(1)免疫组化方法:模型组大鼠肝组织随着给药时间的延长,NF-κB p65蛋白在肝细胞核表达逐渐增多(P<0.01);对照组肝细胞胞核未见NF-κB p65蛋白表达(P>0.05)。(2)RT-PCR结果显示:模型组NF-κB mRNA随着给药时间的延长表达逐渐增加,12h达到最高值,24h较12h有所下降,各时间点比较差别有统计学意义(P<0.01);对照组NF-κB mRNA各时间点表达无明显差别(P>0.05),在4h、8h、12h、24h,NF-κB mRNA表达模型组均明显高于对照组(P<0.05)。
7肝组织NIK检测结果:(1)免疫组化方法:模型组NIK蛋白主要在肝细胞胞浆表达,各时间点比较差别无统计学意义(P>0.05);对照组肝细胞胞浆NIK蛋白未见表达(P>0.05)。(2)RT-PCR结果显示:模型组NIK mRNA各时间点表达差别无统计学意义(P>0.05);对照组NIK mRNA各时间点表达差别无统计学意义(P>0.05),各时间点NIK mRNA表达模型组均明显高于对照组(P<0.05)。
8 IRE1α与Caspase-12、细胞凋亡率的相关性分析及Caspase-12与肝细胞凋亡率的相关性分析:
IRE1α与Caspase-12、细胞凋亡率均成正相关(r= 0.733,P=0.000 ;r= 0.715,P=0.000)。Caspase-12与肝细胞凋亡率成正相关:(r= 0.586,P=0.001)
9 NF-κB p65与细胞凋亡率、IRE1α、NIK的相关性分析:
NF-κB p65与细胞凋亡率、IRE1α均成正相关(r=0.515 ,P=0.004;r= 0.763,P=0.000);NF-κB p65与NIK无相关性(P>0.05)。
结论:
1.利用D-GalN联合LPS制造大鼠暴发性肝衰竭模型,从形态学、细胞学以及基因水平证实了肝细胞凋亡在暴发性肝衰竭病理过程中起着重要作用。
2.在实验性暴发性肝衰竭中,随着肝脏病理损伤的加重肝组织中IRE1α、Caspase-12表达增加,IRE1α与Caspase-12、细胞凋亡率以及Caspase-12与细胞凋亡率均呈明显正相关,提示内质网应激参与了暴发性肝衰竭肝细胞凋亡的发生发展,IRE1α/ TRAF2/ Caspase-12介导的信号转导通路是内质网应激导致暴发性肝衰竭中肝细胞凋亡的重要机制之一。
3.在实验性暴发性肝衰竭中,随着肝脏病理损伤的加重肝组织中NF-κB表达增加,且与肝细胞凋亡率呈明显正相关,从细胞、蛋白、基因水平证实NF-κB在暴发性肝衰竭肝细胞凋亡中起重要作用。
4.在实验性暴发性肝衰竭肝组织中NF-κB p65与IRE1α呈明显正相关,但与NIK之间无相关关系,提示NF-κB可能在内质网应激信号转导通路中起重要作用,但在IRE1α/TRAF2/NF-κB这一信号转导通路中,连接TRAF2与NF-κB的信号转录分子NIK对NF-κB p65无明确调控作用。
Objective: Fulminant hepatic failure(FHF) is a syndrome with the characteristic of severe liver function damage caused by a great quantity of hepatocellular necrosis resulted from many reasons. The characteristics of FHF are acute onset, rapid progression, many complications, high mortality and poor prognosis. It is currently the hot and tough issue at home and abroad. Its pathogenesis is complex and has not been fully clarified so far. The current studies suggest that FHF is closely related to hepatocellular apoptosis. But the mechanism has not been known well. Broadly speaking, there are three pathways in apoptosis: death receptor pathway, mitochondrion pathway and endoplasmic reticulum pathway. Of which, endoplasmic reticulum stress(ERS) mediated apoptosis pathway recently discovered is related to many liver diseases, and it is a hot point of domestic and international research. Endoplasmic reticulum(ER) is the biggest membrane network structure in cell. Its functions are the synthesis and processing of proteins so as to be involved in metabolism and cell signal processing. There are many reasons for physiological dysfunction , the imbalance of Ca2+ and the accumulation of misfolded and unfolded proteins in the ER. Such subcellular pathological phenomena are called ERS. It is an important mechanism by which the misfolded proteins return to the correct conformation and to be further processed. ERS is a self-protection mechanism for cells, but too much and continuous ERS will harm cells even cause apoptosis. The deposition of a large number of membrane proteins induces ER overload response(EOR),and nuclear factorκB (NF-κB) is activated during ERS. Inositol requiring 1( IRE1) play a central role in the EOR pathway.IRE1 is invovled in all the ERS stages such as adpatation , alert, apoptosis as an ER transmembrane protein .In ERS,activated IRE1s are able to recruit tumor necrosis factor receptor associated factor 2(TRAF2)to be IRE1-TRAF2 complex , by which NF-κB is activated.NF-κB as a multiway regulation nuclear transcription factor exists widely in cells. It is closely related to apoptosis .It is involved in transcriptional regulation of apoptosis-related genes and plays a dual role in apoptosis inhibition and apoptosis promotion. Activation of NF-κB is reported to be mediated via TRAF2,another study demonstrated that activation of NF-κB was also reported to be mediated via NIK and activation of NF-κB inducing kinase(NIK) was reported to be mediated via TRAF2,so activation of NF-κB is supposed to be through the pathway IRE1/TRAF2/ NIK/ NF-κB. The family of cysteine aspirate proteases,the so called caspases,are critical mediators of programmed cell death. Caspase-12 as a member of the family of cysteine aspirate proteases is localized to the ER and activated via ERS, but not via membrane- or mitochondrial-targeted apoptotic signals. Recently, it has been demonstrated that apoptosis mediated via ERS is related to many kinds of diseases such as hepatitis B virus ,nonalcoholic fatty liver disease, Alzheimer's disease ,diabetes and so on. But there is still little research on how hepatocellular apoptosis is mediated via ERS in FHF.
In this research, Wistar rats were administered with D-GalN and LPS to be FHF animal model, and the control group were administered with the same volumetric saline simultaneously. Immunohistochemistry,reverse transcriptase polymerase chain reaction (RT-PCR) methods were used to detect the expressions of Caspase-12, IRE1, NF-κB, NIK in the two groups. Hematoxylin-eosin (HE) staining, flow cytometry (FCM) were used to evaluate hepatocellular apoptosis in the two groups. The relationships between IRE1 expression and hepatocellular apoptosis, Caspase-12 were analized respectively,and the relationship between Caspase-12 expression and hepatocellular apoptosis was analized, the effect and mechanism of ERS on FHF were eventually discussed .The relationships between NF-κB activation expression and hepatocellular apoptosis,IRE1,NIK were analized respectively,the effect and mechanism of NF-κB activated via IRE1-mediated ERS pathway, were eventually discussed . These discussions may be good for clarifying the pathogenesis of FHF.
Methods:
1 study objects: 60 masculinity depuratory Wistar rats weighted 180-200g were randomly divided into model group 30 and PDTC group 30. Fulminant hepatic failure model was manufactured by peritoneal injection of D-galactosamine 800 mg/kg,afterward intradermally injection of LPS 10μg/kg in the model group. The control group were administered with the same volumetric saline simultaneously.
2 Sample collection and conservation: 6 rats in model group and control group respectively were sacrificed at 2,4,8,12,24 hours after injection by femoral vein exanguinate. Adequate liver tissue was respectively fixed by 10% neutral formaldehyde solution,70% ethanol. Some of the liver tissue was freezed in liquid nitrogen immediately.
3 Routine HE staining of hepatic tissue: Liver histopathology change was observed under the light microscope.
4.Immunohistochemistry staining of hepatic tissue: The expressions of Caspase-12, IRE1, NF-κBp65 and NIK proteins were detected by immunohistochemistry PV method.
5 The expression of Caspase-12, IRE1, NF-κB and NIK mRNA were detected by RT-PCR in the two groups.
6 The apoptotic rate was detected by flow cytometry method.
7 Statistically analysis: SPSS 13.0 statistical software was used for statistical analysis,data were analyzed by ANOVA, grade and rank and Spearman's rank correlation test.
Results:
1 Observation of rat general state of health: General state of health was aggravated gradually with administration time in model group. Rats became hydroposia decrease,horripilation,downcast,reaction dullness and drowsiness with the time. But in control group, general state of health was good.
2 Liver tissues morphology general observation: (1) Liver tissues showed hyperaemia,bleeding point,partly congestion and necrosis in model group. The fragility of hepatic tissue increased and there was massive necrosis in the model group with the time. Liver tissue in control group was normal, bright red and soft. (2) Hepatic tissue HE staining: The pathological change was aggravated gradually with the time in model group. Hepatocyte edema and acidophilia,apoptotic bodies,inflammatory cell infiltration,liver tissue hemorrhage,hepatocellular necrosis and hepatic lobules disorganization were discovered in liver tissue under light microscope. In control group, hepatic tissue was normal.
3 Changing of hepatocyte apoptosis: The apoptotic rate detected by FCM exhibited an upward trend of apoptotic hypatocytes with the time in model group(P<0.05). In control group, hepatocellular apoptotic rate was low(P>0.05), and less than the model group at 4,8,12,24 hours respectively(P <0.05).
4 Caspase-12 protein and mRNA expression in liver tissue in the two groups: (1) The expression of Caspase-12 protein: The positive staining cell is mainly hepatocyte in model group,and most cytoplasm staining,increased with time. And the difference had statistical significance (P<0.01). The expression of Caspase-12 protein was not discovered in liver tissue in control group.(2) The expression of Caspase-12 mRNA: In model group, it increased with time(P<0.01). There was a peak at 8 hour after admistration and then decreased gradually.In control group , there was weak expression of Caspase-12 mRNA and the expressin of Caspase-12 mRNA were less than the model group at 4,8,12,24 hours respectively(P <0.05).
5 IRE1αprotein and mRNA expression of hepatic tissue in the two groups: (1) The expression of IRE1αprotein: The positive staining cell is mainly hepatocyte in model group,and most cytoplasm staining,increased with time (P<0.01). There was no expression of IRE1αprotein of liver tissue in control group.(2) The expression of IRE1αmRNA: In model group, it increased with time gradually(P<0.01). In control group , there was a little expressin of IRE1αmRNA . The expressin of IRE1αmRNA were less than the model group(P <0.05).
6 NF-κB p65 protein and mRNA expression in liver tissue in the two groups: (1) The expression of NF-κB p65 protein: The positive staining cell was mainly hepatocyte in model group,and most nuclear staining,increased with time,and was strongly positive at 12 hour, then decreased at 24 hour. The expression of NF-κB p65 protein in control group was not discovered and less than the control group (P<0.05). (2)The expression of NF-κB mRNA: In model group, it increased after administration and peaked at 12 hour,then gradually decresed(P<0.01). In control group, liver tissue showed less NF-κB mRNA expression compared with the model group at 2, 4,8,12,24 hours respectively (P<0.05).
7 NIK protein and mRNA expression of liver tissue in the two groups: (1) The expression of NIK protein: The positive staining cell was mainly hepatocyte in model group,and most cytoplasm staining,And the difference had no statistical significance (P>0.05). The expression of NIK protein was not discovered in liver tissue in control group. (2) The expression of NIK mRNA: In model group liver tissue showed more NIK mRNA expression compared with the model group (P<0.05).But there were no difference at different time points in model group or in control group (P>0.05).
8 The relationships of IRE1αand hepatocellular apoptotic rate ,Caspase-12,and the relationship of Caspase-12 and hepatocellular apoptotic rate in experimental fulminant hepatic failure:There were positive correlations between IRE1αand Caspase-12,hepatocellular apoptotic rate(r= 0.733, P=0.000;r= 0.715, P=0.000). There was positive correlation between Caspase-12 and hepatocellular apoptotic rate(r= 0.586, P=0.001).
9 The relationship of NF-κB and hepatocellular apoptotic rate ,IRE1α,NIK in experimental fulminant hepatic failure:There were positive correlations between NF-κB and hepatocellular apoptotic rate,IRE1α(r=0.515 , P=0.004; r= 0.763, P=0.000).There was no correlation between NF-κB and NIK(P>0.05).
Conclusions:
1 The rat model induced by D-GalN+LPS can reflect the pathological changes of fulminant hepatic failure. Hepatocellular apoptosis was observed morphologically,cytologically and from gene level. It's confirmed that hepatocyte apoptosis plays an important role in the progression of FHF.
2 In experimental FHF, there were increased expressions of IRE1α, Caspase-12 with time and there were positive correlations between IRE1αand Caspase-12,hepatocellular apoptotic rate. There was positive correlation between Caspase-12 and hepatocellular apoptotic rate.So it is confirmed that ERS play an important rol in FHF and the IRE1α/ TRAF2/ Caspase-12 signal transduction pathway may be an important mechanism for ERS inducing hepatocellular apoptosis.
3 In experimental FHF,there was increased expression of NF-κB with time and there was positive correlation between NF-κB and hepatocellular apoptotic rate. So it is confirmed that NF-κB may play an important role in FHF hepatocellular apoptosis.
4 In experimental FHF,there was positive correlation between NF-κB p65 and IRE1α,there was no correlation between NF-κB p65 and NIK. So it is confirmed that NF-κB may play an important role in ERS , but NIK may be not a connection molecule between TRAF2 and NF-κB p65 in the IRE1α/ TRAF2/ NF-κB signal transduction pathway.
目前研究认为,FHF与肝细胞凋亡密切相关,但其具体作用机制尚不清楚。内质网应激是除死亡受体活化和线粒体损伤外又一条新的介导细胞凋亡的信号传导通路,与许多肝脏疾病相关,是目前国内外研究的热点。
内质网(endoplasmic reticulum,ER)是细胞内最大的膜网络结构,具有合成、加工蛋白参与代谢和细胞信号处理功能。内质网应激(endoplasmic reticulum stress,ERS)是指由于某种原因导致细胞内质网生理功能发生紊乱,钙稳态失衡,错误折叠或未折叠的蛋白质在内质网腔内聚集的一种亚细胞器的病理状态,是使错误的蛋白质恢复正确构象并能够进一步加工所必需的步骤,是一种细胞自我保护的机制。但是过强或长时间的ERS则可能导致细胞凋亡。内质网应激反应过程中,当大量膜蛋白在内质网沉积时,可激活内质网超负荷反应(ER overload response,EOR),NF-κB(nuclear factorκB)被活化,在EOR信号通路中需肌醇蛋白1(inositol requiring 1,IRE1)处于中心地位,IRE1是内质网跨膜蛋白,参与ERS的适应、警戒、凋亡三个阶段,ERS时IRE1活化,聚集肿瘤坏死因子受体相关因子2(tumor necrosis factor receptor associated factor 2, TRAF2),引起NF-κB活化。NF-κB是一种广泛存在于细胞中具有多向性调节作用的核转录因子,与细胞凋亡关系密切,其参与多种凋亡相关基因的转录调控,具有抑制细胞凋亡和促进细胞凋亡的双重作用。而且有研究表明TRAF2可通过激活NF-κB诱导激酶(NF-κB inducing kinase, NIK)激活NF-κB。Caspase-12是内质网膜结合蛋白,能够且仅被ERS介导的凋亡活化。近年来发现ERS介导的细胞凋亡与乙型肝炎、非酒精性脂肪性肝病、阿尔茨海默病和糖尿病等密切相关,而在暴发性肝衰竭肝细胞凋亡中的作用如何报道较少。本研究应用D-氨基半乳糖( D-galactosamine,D-GalN )和脂多糖(lipopolysaccharide,LPS)制造大鼠暴发性肝衰竭模型,对照组同步注射同样体积的生理盐水进行比较。通过采用免疫组化及反转录多聚酶链反应(reverse transcriptase polymerase chain reaction,RT-PCR)方法检测两组肝组织Caspase-12、IRE1、NF-κB、NIK表达情况,采用常规苏木素-伊红(hematoxylin-eosin,HE)染色,流式细胞学技术(flow cytometry,FCM)观察肝细胞凋亡情况,分析内质网跨膜蛋白IRE1与肝细胞凋亡程度、Caspase-12的相互关系及Caspase-12与肝细胞凋亡程度的相互关系,探讨内质网应激在暴发性肝衰竭肝细胞凋亡中的作用及机制;分析NF-κB活性与肝细胞凋亡程度、IRE1及NIK的关系,探讨IRE1介导NF-κB激活的信号转导通路在暴发性肝衰竭发病机制中的作用,为阐明暴发性肝衰竭的发病机制和防治提供理论依据。
方法: 1研究对象:雄性Wistar大鼠60只,清洁级,体重180-200g。大鼠禁食24小时后,随机分为2组,即模型组和对照组,每组30只。模型组腹腔注射D-GalN800mg/kg,之后皮内注射LPS10ug/kg制造暴发性肝衰竭模型,对照组注射同样体积的生理盐水。2标本采集和保存:从对照组、模型组各取6只分别于给D-GalN +LPS后2、4、8、12、24小时行股静脉放血处死,立即剖腹取出肝脏,取适量肝组织分别固定于10%中性甲醛液、70%乙醇中,部分肝组织经液氮速冻后存放于-80℃冰箱。3 HE染色光镜下观察肝组织病理学变化。4采用免疫组织化学pv法检测肝组织中Caspase-12、IRE1、NF-κB p65、NIK蛋白表达情况。5采用RT-PCR方法检测肝组织中Caspase-12、IRE1、NF-κB p65、NIKmRNA的表达情况。6应用流式细胞学技术测定肝细胞凋亡率(apoptotic rate,AR)。7统计学处理:应用spss13.0统计软件进行统计学分析,采用方差分析,秩和检验和Spearman秩相关分析对数据进行统计。
结果:
1一般状况观察:模型组大鼠随着给药时间的延长出现活动减少、反应迟钝、毛发竖立、嗜睡、昏迷等情况。对照组大鼠一般状况较好。
2肝组织形态学观察:(1)大体标本观察:模型组大鼠肝脏随着给药时间的延长出现充血、出血点及淤血、淤斑逐渐增多,到24小时肝组织呈暗红色,大片出血,淤血严重。对照组大鼠肝脏基本正常。(2)HE染色:模型组大鼠肝组织病理显示给药后2小时肝小叶完整,肝索排列规则;4小时可见肝细胞嗜酸性变及凋亡小体,少量炎细胞浸润;8小时可见炎细胞浸润及多量凋亡小体;12小时肝细胞片状坏死,可见炎细胞浸润,多量凋亡肝细胞;24小时肝组织大片坏死,出血严重,残留散在肝细胞及凋亡小体。对照组大鼠肝组织基本正常。
3细胞凋亡的变化:流式细胞学技术检测细胞凋亡率结果显示在模型组肝细胞凋亡率随着给药时间延长呈现上升趋势(P <0.05);对照组肝细胞凋亡率较低(P>0.05),在4h、8h、12h、24h较模型组细胞凋亡率减少(P <0.05)。
4肝组织Caspase-12检测结果:(1)免疫组化方法:模型组Caspase-12蛋白主要表达在肝细胞浆,随着给药时间的延长表达增多(P<0.01);对照组肝细胞胞浆Caspase-12蛋白未见明显表达。(2)RT-PCR结果显示:模型组Caspase-12 mRNA表达随着给药时间的延长而增加,8h达高峰,以后逐渐下降,各时间点差别有统计学意义(P<0.01),对照组Caspase-12 mRNA各个时间点弱表达(P>0.05),在4h、8h、12h、24h,模型组Caspase-12 mRNA表达均明显高于对照组(P<0.05)。
5肝组织IRE1α检测结果:(1)免疫组化方法:模型组IRE1α蛋白主要在肝细胞浆表达,随着给药时间的延长,IRE1α蛋白表达明显增多(P<0.01);对照组肝细胞胞质IRE1α蛋白未见明显表达。(2)RT-PCR结果显示:模型组随着给药时间的延长IRE1αmRNA表达增高(P<0.01);对照组IRE1αmRNA各个时间点弱表达(P>0.05),各时间点模型组IRE1αmRNA表达均明显高于对照组(P<0.05)。
6肝组织NF-κBp65检测结果:(1)免疫组化方法:模型组大鼠肝组织随着给药时间的延长,NF-κB p65蛋白在肝细胞核表达逐渐增多(P<0.01);对照组肝细胞胞核未见NF-κB p65蛋白表达(P>0.05)。(2)RT-PCR结果显示:模型组NF-κB mRNA随着给药时间的延长表达逐渐增加,12h达到最高值,24h较12h有所下降,各时间点比较差别有统计学意义(P<0.01);对照组NF-κB mRNA各时间点表达无明显差别(P>0.05),在4h、8h、12h、24h,NF-κB mRNA表达模型组均明显高于对照组(P<0.05)。
7肝组织NIK检测结果:(1)免疫组化方法:模型组NIK蛋白主要在肝细胞胞浆表达,各时间点比较差别无统计学意义(P>0.05);对照组肝细胞胞浆NIK蛋白未见表达(P>0.05)。(2)RT-PCR结果显示:模型组NIK mRNA各时间点表达差别无统计学意义(P>0.05);对照组NIK mRNA各时间点表达差别无统计学意义(P>0.05),各时间点NIK mRNA表达模型组均明显高于对照组(P<0.05)。
8 IRE1α与Caspase-12、细胞凋亡率的相关性分析及Caspase-12与肝细胞凋亡率的相关性分析:
IRE1α与Caspase-12、细胞凋亡率均成正相关(r= 0.733,P=0.000 ;r= 0.715,P=0.000)。Caspase-12与肝细胞凋亡率成正相关:(r= 0.586,P=0.001)
9 NF-κB p65与细胞凋亡率、IRE1α、NIK的相关性分析:
NF-κB p65与细胞凋亡率、IRE1α均成正相关(r=0.515 ,P=0.004;r= 0.763,P=0.000);NF-κB p65与NIK无相关性(P>0.05)。
结论:
1.利用D-GalN联合LPS制造大鼠暴发性肝衰竭模型,从形态学、细胞学以及基因水平证实了肝细胞凋亡在暴发性肝衰竭病理过程中起着重要作用。
2.在实验性暴发性肝衰竭中,随着肝脏病理损伤的加重肝组织中IRE1α、Caspase-12表达增加,IRE1α与Caspase-12、细胞凋亡率以及Caspase-12与细胞凋亡率均呈明显正相关,提示内质网应激参与了暴发性肝衰竭肝细胞凋亡的发生发展,IRE1α/ TRAF2/ Caspase-12介导的信号转导通路是内质网应激导致暴发性肝衰竭中肝细胞凋亡的重要机制之一。
3.在实验性暴发性肝衰竭中,随着肝脏病理损伤的加重肝组织中NF-κB表达增加,且与肝细胞凋亡率呈明显正相关,从细胞、蛋白、基因水平证实NF-κB在暴发性肝衰竭肝细胞凋亡中起重要作用。
4.在实验性暴发性肝衰竭肝组织中NF-κB p65与IRE1α呈明显正相关,但与NIK之间无相关关系,提示NF-κB可能在内质网应激信号转导通路中起重要作用,但在IRE1α/TRAF2/NF-κB这一信号转导通路中,连接TRAF2与NF-κB的信号转录分子NIK对NF-κB p65无明确调控作用。
Objective: Fulminant hepatic failure(FHF) is a syndrome with the characteristic of severe liver function damage caused by a great quantity of hepatocellular necrosis resulted from many reasons. The characteristics of FHF are acute onset, rapid progression, many complications, high mortality and poor prognosis. It is currently the hot and tough issue at home and abroad. Its pathogenesis is complex and has not been fully clarified so far. The current studies suggest that FHF is closely related to hepatocellular apoptosis. But the mechanism has not been known well. Broadly speaking, there are three pathways in apoptosis: death receptor pathway, mitochondrion pathway and endoplasmic reticulum pathway. Of which, endoplasmic reticulum stress(ERS) mediated apoptosis pathway recently discovered is related to many liver diseases, and it is a hot point of domestic and international research. Endoplasmic reticulum(ER) is the biggest membrane network structure in cell. Its functions are the synthesis and processing of proteins so as to be involved in metabolism and cell signal processing. There are many reasons for physiological dysfunction , the imbalance of Ca2+ and the accumulation of misfolded and unfolded proteins in the ER. Such subcellular pathological phenomena are called ERS. It is an important mechanism by which the misfolded proteins return to the correct conformation and to be further processed. ERS is a self-protection mechanism for cells, but too much and continuous ERS will harm cells even cause apoptosis. The deposition of a large number of membrane proteins induces ER overload response(EOR),and nuclear factorκB (NF-κB) is activated during ERS. Inositol requiring 1( IRE1) play a central role in the EOR pathway.IRE1 is invovled in all the ERS stages such as adpatation , alert, apoptosis as an ER transmembrane protein .In ERS,activated IRE1s are able to recruit tumor necrosis factor receptor associated factor 2(TRAF2)to be IRE1-TRAF2 complex , by which NF-κB is activated.NF-κB as a multiway regulation nuclear transcription factor exists widely in cells. It is closely related to apoptosis .It is involved in transcriptional regulation of apoptosis-related genes and plays a dual role in apoptosis inhibition and apoptosis promotion. Activation of NF-κB is reported to be mediated via TRAF2,another study demonstrated that activation of NF-κB was also reported to be mediated via NIK and activation of NF-κB inducing kinase(NIK) was reported to be mediated via TRAF2,so activation of NF-κB is supposed to be through the pathway IRE1/TRAF2/ NIK/ NF-κB. The family of cysteine aspirate proteases,the so called caspases,are critical mediators of programmed cell death. Caspase-12 as a member of the family of cysteine aspirate proteases is localized to the ER and activated via ERS, but not via membrane- or mitochondrial-targeted apoptotic signals. Recently, it has been demonstrated that apoptosis mediated via ERS is related to many kinds of diseases such as hepatitis B virus ,nonalcoholic fatty liver disease, Alzheimer's disease ,diabetes and so on. But there is still little research on how hepatocellular apoptosis is mediated via ERS in FHF.
In this research, Wistar rats were administered with D-GalN and LPS to be FHF animal model, and the control group were administered with the same volumetric saline simultaneously. Immunohistochemistry,reverse transcriptase polymerase chain reaction (RT-PCR) methods were used to detect the expressions of Caspase-12, IRE1, NF-κB, NIK in the two groups. Hematoxylin-eosin (HE) staining, flow cytometry (FCM) were used to evaluate hepatocellular apoptosis in the two groups. The relationships between IRE1 expression and hepatocellular apoptosis, Caspase-12 were analized respectively,and the relationship between Caspase-12 expression and hepatocellular apoptosis was analized, the effect and mechanism of ERS on FHF were eventually discussed .The relationships between NF-κB activation expression and hepatocellular apoptosis,IRE1,NIK were analized respectively,the effect and mechanism of NF-κB activated via IRE1-mediated ERS pathway, were eventually discussed . These discussions may be good for clarifying the pathogenesis of FHF.
Methods:
1 study objects: 60 masculinity depuratory Wistar rats weighted 180-200g were randomly divided into model group 30 and PDTC group 30. Fulminant hepatic failure model was manufactured by peritoneal injection of D-galactosamine 800 mg/kg,afterward intradermally injection of LPS 10μg/kg in the model group. The control group were administered with the same volumetric saline simultaneously.
2 Sample collection and conservation: 6 rats in model group and control group respectively were sacrificed at 2,4,8,12,24 hours after injection by femoral vein exanguinate. Adequate liver tissue was respectively fixed by 10% neutral formaldehyde solution,70% ethanol. Some of the liver tissue was freezed in liquid nitrogen immediately.
3 Routine HE staining of hepatic tissue: Liver histopathology change was observed under the light microscope.
4.Immunohistochemistry staining of hepatic tissue: The expressions of Caspase-12, IRE1, NF-κBp65 and NIK proteins were detected by immunohistochemistry PV method.
5 The expression of Caspase-12, IRE1, NF-κB and NIK mRNA were detected by RT-PCR in the two groups.
6 The apoptotic rate was detected by flow cytometry method.
7 Statistically analysis: SPSS 13.0 statistical software was used for statistical analysis,data were analyzed by ANOVA, grade and rank and Spearman's rank correlation test.
Results:
1 Observation of rat general state of health: General state of health was aggravated gradually with administration time in model group. Rats became hydroposia decrease,horripilation,downcast,reaction dullness and drowsiness with the time. But in control group, general state of health was good.
2 Liver tissues morphology general observation: (1) Liver tissues showed hyperaemia,bleeding point,partly congestion and necrosis in model group. The fragility of hepatic tissue increased and there was massive necrosis in the model group with the time. Liver tissue in control group was normal, bright red and soft. (2) Hepatic tissue HE staining: The pathological change was aggravated gradually with the time in model group. Hepatocyte edema and acidophilia,apoptotic bodies,inflammatory cell infiltration,liver tissue hemorrhage,hepatocellular necrosis and hepatic lobules disorganization were discovered in liver tissue under light microscope. In control group, hepatic tissue was normal.
3 Changing of hepatocyte apoptosis: The apoptotic rate detected by FCM exhibited an upward trend of apoptotic hypatocytes with the time in model group(P<0.05). In control group, hepatocellular apoptotic rate was low(P>0.05), and less than the model group at 4,8,12,24 hours respectively(P <0.05).
4 Caspase-12 protein and mRNA expression in liver tissue in the two groups: (1) The expression of Caspase-12 protein: The positive staining cell is mainly hepatocyte in model group,and most cytoplasm staining,increased with time. And the difference had statistical significance (P<0.01). The expression of Caspase-12 protein was not discovered in liver tissue in control group.(2) The expression of Caspase-12 mRNA: In model group, it increased with time(P<0.01). There was a peak at 8 hour after admistration and then decreased gradually.In control group , there was weak expression of Caspase-12 mRNA and the expressin of Caspase-12 mRNA were less than the model group at 4,8,12,24 hours respectively(P <0.05).
5 IRE1αprotein and mRNA expression of hepatic tissue in the two groups: (1) The expression of IRE1αprotein: The positive staining cell is mainly hepatocyte in model group,and most cytoplasm staining,increased with time (P<0.01). There was no expression of IRE1αprotein of liver tissue in control group.(2) The expression of IRE1αmRNA: In model group, it increased with time gradually(P<0.01). In control group , there was a little expressin of IRE1αmRNA . The expressin of IRE1αmRNA were less than the model group(P <0.05).
6 NF-κB p65 protein and mRNA expression in liver tissue in the two groups: (1) The expression of NF-κB p65 protein: The positive staining cell was mainly hepatocyte in model group,and most nuclear staining,increased with time,and was strongly positive at 12 hour, then decreased at 24 hour. The expression of NF-κB p65 protein in control group was not discovered and less than the control group (P<0.05). (2)The expression of NF-κB mRNA: In model group, it increased after administration and peaked at 12 hour,then gradually decresed(P<0.01). In control group, liver tissue showed less NF-κB mRNA expression compared with the model group at 2, 4,8,12,24 hours respectively (P<0.05).
7 NIK protein and mRNA expression of liver tissue in the two groups: (1) The expression of NIK protein: The positive staining cell was mainly hepatocyte in model group,and most cytoplasm staining,And the difference had no statistical significance (P>0.05). The expression of NIK protein was not discovered in liver tissue in control group. (2) The expression of NIK mRNA: In model group liver tissue showed more NIK mRNA expression compared with the model group (P<0.05).But there were no difference at different time points in model group or in control group (P>0.05).
8 The relationships of IRE1αand hepatocellular apoptotic rate ,Caspase-12,and the relationship of Caspase-12 and hepatocellular apoptotic rate in experimental fulminant hepatic failure:There were positive correlations between IRE1αand Caspase-12,hepatocellular apoptotic rate(r= 0.733, P=0.000;r= 0.715, P=0.000). There was positive correlation between Caspase-12 and hepatocellular apoptotic rate(r= 0.586, P=0.001).
9 The relationship of NF-κB and hepatocellular apoptotic rate ,IRE1α,NIK in experimental fulminant hepatic failure:There were positive correlations between NF-κB and hepatocellular apoptotic rate,IRE1α(r=0.515 , P=0.004; r= 0.763, P=0.000).There was no correlation between NF-κB and NIK(P>0.05).
Conclusions:
1 The rat model induced by D-GalN+LPS can reflect the pathological changes of fulminant hepatic failure. Hepatocellular apoptosis was observed morphologically,cytologically and from gene level. It's confirmed that hepatocyte apoptosis plays an important role in the progression of FHF.
2 In experimental FHF, there were increased expressions of IRE1α, Caspase-12 with time and there were positive correlations between IRE1αand Caspase-12,hepatocellular apoptotic rate. There was positive correlation between Caspase-12 and hepatocellular apoptotic rate.So it is confirmed that ERS play an important rol in FHF and the IRE1α/ TRAF2/ Caspase-12 signal transduction pathway may be an important mechanism for ERS inducing hepatocellular apoptosis.
3 In experimental FHF,there was increased expression of NF-κB with time and there was positive correlation between NF-κB and hepatocellular apoptotic rate. So it is confirmed that NF-κB may play an important role in FHF hepatocellular apoptosis.
4 In experimental FHF,there was positive correlation between NF-κB p65 and IRE1α,there was no correlation between NF-κB p65 and NIK. So it is confirmed that NF-κB may play an important role in ERS , but NIK may be not a connection molecule between TRAF2 and NF-κB p65 in the IRE1α/ TRAF2/ NF-κB signal transduction pathway.
引文
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21 Shakhov AN, Collart MA, Vassalli P, et al. Kappa B-type enhancers are involved in lipopolysaccharide-mediated transcriptional activation of the tumor necrosis factor alpha gene in primary macrophages. J Exp Med, 1990,171:35-47
22 Fujioka S, Schmidt C, Sclabas GM, et al. Stabilization of p53 is a novel mechanism for proapoptotic function of NF-kappaB. J Biol Chem, 2004,279:27549-27559
23曹晓伟,晿浩. Cyclin D1和NF-κB在5-Fu诱导肝癌细胞凋亡过程中的表达.实用癌症杂志, 2005:28-30
24 Tai DI, Tsai SL, Chang YH, et al. Constitutive activation of nuclear factor kappaB in hepatocellular carcinoma. Cancer, 2000,89:2274-2281
25 Nanji AA, Jokelainen K, Rahemtulla A, et al. Activation of nuclear factor kappa B and cytokine imbalance in experimental alcoholic liver disease in the rat. Hepatology, 1999,30:934-943
26 Cavin LG, Venkatraman M, Factor VM, et al. Regulation of alpha-fetoprotein by nuclear factor-kappaB protects hepatocytes from tumor necrosis factor-alpha cytotoxicity during fetal liver development and hepatic oncogenesis. Cancer Res, 2004,64:7030-7038
27崔香丹,金武丕,李香.大鼠酒精性肝病细胞凋亡中Caspase-3、NF-κB的表达.陕西医学杂志, 2009:273-275.
28廖达林,张志坚,林克荣,等.非酒精性脂肪性肝病大鼠肝细胞凋亡及核因子κB的活化.中国临床康复, 2006:97-99
29 Nemeth ZH, Deitch EA, Lu Q, et al. NHE blockade inhibits chemokine production and NF-kappaB activation in immunostimulated endothelial cells. Am J Physiol Cell Physiol, 2002,283:C396-403
30 Matsuda T, Almasan A, Tomita M, et al. Dengue virus-induced apoptosis in hepatic cells is partly mediated by Apo2 ligand/tumour necrosis factor-related apoptosis-inducing ligand. J Gen Virol, 2005,86:1055-1065
31 Yamazaki S, Muta T, Takeshige K. A novel IkappaB protein, IkappaB-zeta, induced by proinflammatory stimuli, negatively regulates nuclear factor-kappaB in the nuclei. J Biol Chem, 2001,276:27657-27662
32 Pahl HL, Baeuerle PA. The ER-overload response: activation of NF-kappa B. Trends Biochem Sci, 1997,22:63-67
33 Pahl HL, Baeuerle PA. A novel signal transduction pathway from the endoplasmic reticulum to the nucleus is mediated by transcription factorNF-kappa B. EMBO J, 1995,14:2580-2588.
34 Kaneko M, Niinuma Y, Nomura Y. Activation signal of nuclear factor-kappa B in response to endoplasmic reticulum stress is transduced via IRE1 and tumor necrosis factor receptor-associated factor 2. Biol Pharm Bull, 2003,26:931-935
35 Hu P, Han Z, Couvillon AD, et al. Autocrine tumor necrosis factor alpha links endoplasmic reticulum stress to the membrane death receptor pathway through IRE1alpha-mediated NF-kappaB activation and down-regulation of TRAF2 expression. Mol Cell Biol, 2006,26:3071-3084
36 Kaneko M, Takahashi T, Niinuma Y, et al. Manganese superoxide dismutase is induced by endoplasmic reticulum stress through IRE1-mediated nuclear factor (NF)-kappaB and AP-1 activation. Biol Pharm Bull, 2004,27:1202-1206
37 Wittwer T, Schmitz ML. NIK and Cot cooperate to trigger NF-kappaB p65 phosphorylation. Biochem Biophys Res Commun, 2008,371:294-297
38 Jijon H, Allard B, Jobin C. NF-kappaB inducing kinase activates NF-kappaB transcriptional activity independently of IkappaB kinase gamma through a p38 MAPK-dependent RelA phosphorylation pathway. Cell Signal, 2004,16:1023-1032
39 Malinin NL, Boldin MP, Kovalenko AV, et al. MAP3K-related kinase involved in NF-kappaB induction by TNF, CD95 and IL-1. Nature, 1997,385:540-544
40 Zarnegar BJ, Wang Y, Mahoney DJ, et al. Noncanonical NF-kappaB activation requires coordinated assembly of a regulatory complex of the adaptors cIAP1, cIAP2, TRAF2 and TRAF3 and the kinase NIK. Nat Immunol, 2008,9:1371-1378
41 Carpentier I, Declercq W, Malinin NL, et al. TRAF2 plays a dual role in NF-kappaB-dependent gene activation by mediating the TNF-induced activation of p38 MAPK and IkappaB kinase pathways. FEBS Lett, 1998,425:195-198
42 Nishina T, Yamaguchi N, Gohda J, et al. NIK is involved in constitutiveactivation of the alternative NF-kappaB pathway and proliferation of pancreatic cancer cells. Biochem Biophys Res Commun, 2009,388:96-101
43 Vaira S, Johnson T, Hirbe AC, et al. RelB is the NF-kappaB subunit downstream of NIK responsible for osteoclast differentiation. Proc Natl Acad Sci U S A, 2008,105:3897-3902
44 Ling L, Cao Z, Goeddel DV. NF-kappaB-inducing kinase activates IKK-alpha by phosphorylation of Ser-176. Proc Natl Acad Sci U S A, 1998,95:3792-3797
1 Boyce M, Yuan J. Cellular response to endoplasmic reticulum stress:a matter of life or death. Cell Death Differ, 2006;13(3):363-373
2 Shibata M, Hattori H, Sasaki T, et al. Activation of caspase-12 by endoplasmic reticulum stress induced by transient middle cerebral artery occlusion in mice. Neuroscience, 2003, 118(2): 491-499
3 Vilatoba M, Eckstein C, Bilbao G, et al. Sodium 4-phenylbutyrate protects against liver ischemia reperfusioninjury by inhibition of endoplasmic reticulum-stress mediated apoptosis. Surgery, 2005, 138(2): 342-351
4 Ma QB, Gao W, Guo YH, et al.Hypoxia/reoxygen inducedendoplasmic reticulum stress in cultured neonatal rat cardiomyocyte. Beijing Da Xue Xue Bao, 2005, 37(4):386-388
5 Li B, Gao B, Ye LB , et al. Hepatitis B virus X protein (HBx) activates ATF6 and IRE1-XBP1 pathways of unfolded protein response. Virus Research, 2007, 124(1-2): 44-49
6 Hanada S, Harada M, Kuen H, et al.Oxidative Stress induces the endoplamic reticulum stress and facilititates inclusion formation in cultured cells. J Hepatol, 2007, 47(1):93-102
7 Kelly E, Greene CM, Carroll TP, et al. Selenoprotein S/SEPS1 modifies endoplasmic reticulum stress in Z variant alpha1-antitrypsin deficiency. J Biol Chem, 2009, 284(25):16891-16897
8 Kondo S, Saito A, Hino S, et al. BBF2H7, a novel transmembrane bZIP transcription factor, is a new type of endoplasmic reticulum stress transducer. Mol Cell Biol , 2007, 27(5):1716-1729
9 Lin JH, Li Han, Yasumura D, et al. IRE1 signaling affects cell fate during the unfolded protein response. Science , 2007, 318(5852):944-949.
10 Cullinan SB, Diehl JA.Coordination of ER and oxidative stress signaling: the PERK/Nrf2 signaling pathway. Int J Biochem Cell Biol, 2006, 38(3):317-332.
11 Kaufman RJ.Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls. Genes Dev, 1999, 13(10):1211-1233
12 Nishitoh H, Matsuzawa A, Tobiume K, et al.ASK1 is essential for endoplasmic reticulum stress-induced neuronal cell death triggered by expanded polyglutamine repeats. Genes Dev, 2002, 16(11):1345-1355
13 Nakagawa T, Zhu H, Morishima N, et al.Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta. Nature. 2000, 403 (6763) :98-103
14 Pavio N, Romano PR, Graczyk TM, et al. Protein synthesis and endoplasmic reticulum stress can be modulated by the hepatitis C virus envelope protein E2 through the eukaryotic initiation factor 2alpha kinasePERK. J Virol , 2003, 77(6): 3578–3585
15 Chan SW, Egan PA. Hepatitis C virus envelope proteins regulate CHOP via induction of the unfolded protein response. FASEB J , 2005, 19(11): 1510–1512
16 Wei YR, Wang D, Topczewski F, et al. Saturated fatty acids induce endoplasmic reticulum stress and apoptosis independently of ceramide in liver cells. Am J Physiol Endocrinol Metab , 2006, 291(2): E275-E281.
17 Pachikian BD, Neyrinck AM, Cani PD, et al. Hepatic steatosis in n-3 fatty acid depleted mice: focus on metabolic alterations related to tissue fatty acid composition. BMC Physiol, 2008, 8:21
18 Rutkowski DT, Wu J, Back SH, et al.UPR pathways combine to prevent hepatic steatosis caused by ER stress-mediated suppression of transcriptional master regulators. Dev Cell, 2008 , 15(6):829-840
19陈璐璐,李凝旭,邓向群.限食对高脂喂养大鼠肝脏内质网应激的影响.中国病理生理杂志, 2008, 24(3): 568–572
20 Ji C, Kaplowitz N. Betaine decreases hyperhomocysteinemia, endoplasmic reticulum stress, and liver injury in alcohol-fed mice. Gastroenterology, 2003, 124(5): 1488–1499
21 Esfandiari F, Villanueva JA, Wong DH, et al. Chronic ethanol feeding and folate deficiency activate hepatic endoplasmic reticulum stress pathway in micropigs. Am J Physiol Gastrointest Liver Physiol, 2005, 289(1): G54–G63
22 Perlmutter DH. The role of autophagy in alpha-1-antitrypsin deficiency: A specific cellular response in genetic diseases associated with aggregation-prone proteins. Autophagy, 2006, 2(4): 258–263
23 Jian BX , Hsieh CH, Chen J. Activation of endoplasmic reticulum stress response following trauma-hemorrhage . Biochim Biophys Acta, 2008, 1782(11) : 621–626
24 Nagy G, Kardon T, Wunderlich L, et al. Acetaminophen induces ER dependent signaling in mouse liver. Arch Biochem Biophys, 2007, 459(2): 273–279
25 Zhou H, Gurley E, Jarujaron S, et al. HIY protease inhibitors activate the unfolded protein response and disrupt lipid metabolism in primary hepatocytes. Am J Physiol, 2006, 291(6): G1071–G1080
26温韬,张海燕,卢静,等.内质网应激在四氯化碳致大鼠急性肝损伤中的作用探讨.胃肠病学和肝病学杂志, 2008, 17(10): 786-789
27姜山,谢青,周惠娟,等.Caspase-12活化在小鼠原代肝细胞凋亡中作用.肝脏, 2006, 11(1):12-14
28 Hiramatsu N, Kasai A, Du S, et al. Rapid, transient induction of ER stress in the liver and kidney after acute exposure to heavy metal: evidence from transgenic sensor mice. FEBS Lett, 2007, 581(10): 2055–2059
2 Yan BZ, Wang W, Chen LY, et al. Role of cathepsin B-mediated apoptosis in fulminant hepatic failure in mice. World J Gastroenterol, 2009,15:1231-1236
3 Guicciardi ME, Gores GJ. Apoptosis: a mechanism of acute and chronic liver injury. Gut, 2005,54:1024-1033.
4王玉梅,冯国和,窦晓光,等.小鼠暴发性肝衰竭肝细胞凋亡形态学及其调控机制.世界华人消化杂志, 2005:2658-2662
5 Canbay A, Friedman S, Gores GJ. Apoptosis: the nexus of liver injury and fibrosis. Hepatology, 2004,39:273-278
6 Takahashi N, Ishizuya T, Mori N. In-vitro preparation of experimental models of hepatitis with D-galactosamine and their modification by liver-repairing factors. Int J Tissue React, 1990,12:263-268
7 Newsome PN, Plevris JN, Nelson LJ, et al. Animal models of fulminant hepatic failure: a critical evaluation. Liver Transpl, 2000,6:21-31
8 Lei K, Davis RJ. JNK phosphorylation of Bim-related members of the Bcl2 family induces Bax-dependent apoptosis. Proc Natl Acad Sci U S A, 2003,100:2432-2437
9 Yoneda T, Imaizumi K, Oono K, et al. Activation of caspase-12, an endoplastic reticulum (ER) resident caspase, through tumor necrosis factor receptor-associated factor 2-dependent mechanism in response to the ER stress. J Biol Chem, 2001,276:13935-13940
10 Mori K, Ma W, Gething MJ, et al. A transmembrane protein with a cdc2+/CDC28-related kinase activity is required for signaling from the ER to the nucleus. Cell, 1993,74:743-756
11 Urano F, Wang X, Bertolotti A, et al. Coupling of stress in the ER toactivation of JNK protein kinases by transmembrane protein kinase IRE1. Science, 2000,287:664-666
12刘芳,邹萍,陈敏,等. Caspase-12短发夹状RNA在内质网应激性凋亡中的作用.中华实验外科杂志, 2005:1568-1570
13 Nakagawa T, Zhu H, Morishima N, et al. Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta. Nature, 2000,403:98-103
14 Ferri KF, Kroemer G. Organelle-specific initiation of cell death pathways. Nat Cell Biol, 2001,3:E255-263
15 Morishima N, Nakanishi K, Takenouchi H, et al. An endoplasmic reticulum stress-specific caspase cascade in apoptosis. Cytochrome c-independent activation of caspase-9 by caspase-12. J Biol Chem, 2002,277:34287-34294
16 Nakagawa T, Yuan J. Cross-talk between two cysteine protease families. Activation of caspase-12 by calpain in apoptosis. J Cell Biol, 2000,150:887-894
17 Rao RV, Peel A, Logvinova A, et al. Coupling endoplasmic reticulum stress to the cell death program: role of the ER chaperone GRP78. FEBS Lett, 2002,514:122-128
18 Xie Q, Khaoustov VI, Chung CC, et al. Effect of tauroursodeoxycholic acid on endoplasmic reticulum stress-induced caspase-12 activation. Hepatology, 2002,36:592-601
19 Karin M, Lin A. NF-kappaB at the crossroads of life and death. Nat Immunol, 2002,3:221-227
20 Pham CG, Bubici C, Zazzeroni F, et al. Ferritin heavy chain upregulation by NF-kappaB inhibits TNFalpha-induced apoptosis by suppressing reactive oxygen species. Cell, 2004,119:529-542
21 Shakhov AN, Collart MA, Vassalli P, et al. Kappa B-type enhancers are involved in lipopolysaccharide-mediated transcriptional activation of the tumor necrosis factor alpha gene in primary macrophages. J Exp Med, 1990,171:35-47
22 Fujioka S, Schmidt C, Sclabas GM, et al. Stabilization of p53 is a novel mechanism for proapoptotic function of NF-kappaB. J Biol Chem, 2004,279:27549-27559
23曹晓伟,晿浩. Cyclin D1和NF-κB在5-Fu诱导肝癌细胞凋亡过程中的表达.实用癌症杂志, 2005:28-30
24 Tai DI, Tsai SL, Chang YH, et al. Constitutive activation of nuclear factor kappaB in hepatocellular carcinoma. Cancer, 2000,89:2274-2281
25 Nanji AA, Jokelainen K, Rahemtulla A, et al. Activation of nuclear factor kappa B and cytokine imbalance in experimental alcoholic liver disease in the rat. Hepatology, 1999,30:934-943
26 Cavin LG, Venkatraman M, Factor VM, et al. Regulation of alpha-fetoprotein by nuclear factor-kappaB protects hepatocytes from tumor necrosis factor-alpha cytotoxicity during fetal liver development and hepatic oncogenesis. Cancer Res, 2004,64:7030-7038
27崔香丹,金武丕,李香.大鼠酒精性肝病细胞凋亡中Caspase-3、NF-κB的表达.陕西医学杂志, 2009:273-275.
28廖达林,张志坚,林克荣,等.非酒精性脂肪性肝病大鼠肝细胞凋亡及核因子κB的活化.中国临床康复, 2006:97-99
29 Nemeth ZH, Deitch EA, Lu Q, et al. NHE blockade inhibits chemokine production and NF-kappaB activation in immunostimulated endothelial cells. Am J Physiol Cell Physiol, 2002,283:C396-403
30 Matsuda T, Almasan A, Tomita M, et al. Dengue virus-induced apoptosis in hepatic cells is partly mediated by Apo2 ligand/tumour necrosis factor-related apoptosis-inducing ligand. J Gen Virol, 2005,86:1055-1065
31 Yamazaki S, Muta T, Takeshige K. A novel IkappaB protein, IkappaB-zeta, induced by proinflammatory stimuli, negatively regulates nuclear factor-kappaB in the nuclei. J Biol Chem, 2001,276:27657-27662
32 Pahl HL, Baeuerle PA. The ER-overload response: activation of NF-kappa B. Trends Biochem Sci, 1997,22:63-67
33 Pahl HL, Baeuerle PA. A novel signal transduction pathway from the endoplasmic reticulum to the nucleus is mediated by transcription factorNF-kappa B. EMBO J, 1995,14:2580-2588.
34 Kaneko M, Niinuma Y, Nomura Y. Activation signal of nuclear factor-kappa B in response to endoplasmic reticulum stress is transduced via IRE1 and tumor necrosis factor receptor-associated factor 2. Biol Pharm Bull, 2003,26:931-935
35 Hu P, Han Z, Couvillon AD, et al. Autocrine tumor necrosis factor alpha links endoplasmic reticulum stress to the membrane death receptor pathway through IRE1alpha-mediated NF-kappaB activation and down-regulation of TRAF2 expression. Mol Cell Biol, 2006,26:3071-3084
36 Kaneko M, Takahashi T, Niinuma Y, et al. Manganese superoxide dismutase is induced by endoplasmic reticulum stress through IRE1-mediated nuclear factor (NF)-kappaB and AP-1 activation. Biol Pharm Bull, 2004,27:1202-1206
37 Wittwer T, Schmitz ML. NIK and Cot cooperate to trigger NF-kappaB p65 phosphorylation. Biochem Biophys Res Commun, 2008,371:294-297
38 Jijon H, Allard B, Jobin C. NF-kappaB inducing kinase activates NF-kappaB transcriptional activity independently of IkappaB kinase gamma through a p38 MAPK-dependent RelA phosphorylation pathway. Cell Signal, 2004,16:1023-1032
39 Malinin NL, Boldin MP, Kovalenko AV, et al. MAP3K-related kinase involved in NF-kappaB induction by TNF, CD95 and IL-1. Nature, 1997,385:540-544
40 Zarnegar BJ, Wang Y, Mahoney DJ, et al. Noncanonical NF-kappaB activation requires coordinated assembly of a regulatory complex of the adaptors cIAP1, cIAP2, TRAF2 and TRAF3 and the kinase NIK. Nat Immunol, 2008,9:1371-1378
41 Carpentier I, Declercq W, Malinin NL, et al. TRAF2 plays a dual role in NF-kappaB-dependent gene activation by mediating the TNF-induced activation of p38 MAPK and IkappaB kinase pathways. FEBS Lett, 1998,425:195-198
42 Nishina T, Yamaguchi N, Gohda J, et al. NIK is involved in constitutiveactivation of the alternative NF-kappaB pathway and proliferation of pancreatic cancer cells. Biochem Biophys Res Commun, 2009,388:96-101
43 Vaira S, Johnson T, Hirbe AC, et al. RelB is the NF-kappaB subunit downstream of NIK responsible for osteoclast differentiation. Proc Natl Acad Sci U S A, 2008,105:3897-3902
44 Ling L, Cao Z, Goeddel DV. NF-kappaB-inducing kinase activates IKK-alpha by phosphorylation of Ser-176. Proc Natl Acad Sci U S A, 1998,95:3792-3797
1 Boyce M, Yuan J. Cellular response to endoplasmic reticulum stress:a matter of life or death. Cell Death Differ, 2006;13(3):363-373
2 Shibata M, Hattori H, Sasaki T, et al. Activation of caspase-12 by endoplasmic reticulum stress induced by transient middle cerebral artery occlusion in mice. Neuroscience, 2003, 118(2): 491-499
3 Vilatoba M, Eckstein C, Bilbao G, et al. Sodium 4-phenylbutyrate protects against liver ischemia reperfusioninjury by inhibition of endoplasmic reticulum-stress mediated apoptosis. Surgery, 2005, 138(2): 342-351
4 Ma QB, Gao W, Guo YH, et al.Hypoxia/reoxygen inducedendoplasmic reticulum stress in cultured neonatal rat cardiomyocyte. Beijing Da Xue Xue Bao, 2005, 37(4):386-388
5 Li B, Gao B, Ye LB , et al. Hepatitis B virus X protein (HBx) activates ATF6 and IRE1-XBP1 pathways of unfolded protein response. Virus Research, 2007, 124(1-2): 44-49
6 Hanada S, Harada M, Kuen H, et al.Oxidative Stress induces the endoplamic reticulum stress and facilititates inclusion formation in cultured cells. J Hepatol, 2007, 47(1):93-102
7 Kelly E, Greene CM, Carroll TP, et al. Selenoprotein S/SEPS1 modifies endoplasmic reticulum stress in Z variant alpha1-antitrypsin deficiency. J Biol Chem, 2009, 284(25):16891-16897
8 Kondo S, Saito A, Hino S, et al. BBF2H7, a novel transmembrane bZIP transcription factor, is a new type of endoplasmic reticulum stress transducer. Mol Cell Biol , 2007, 27(5):1716-1729
9 Lin JH, Li Han, Yasumura D, et al. IRE1 signaling affects cell fate during the unfolded protein response. Science , 2007, 318(5852):944-949.
10 Cullinan SB, Diehl JA.Coordination of ER and oxidative stress signaling: the PERK/Nrf2 signaling pathway. Int J Biochem Cell Biol, 2006, 38(3):317-332.
11 Kaufman RJ.Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls. Genes Dev, 1999, 13(10):1211-1233
12 Nishitoh H, Matsuzawa A, Tobiume K, et al.ASK1 is essential for endoplasmic reticulum stress-induced neuronal cell death triggered by expanded polyglutamine repeats. Genes Dev, 2002, 16(11):1345-1355
13 Nakagawa T, Zhu H, Morishima N, et al.Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta. Nature. 2000, 403 (6763) :98-103
14 Pavio N, Romano PR, Graczyk TM, et al. Protein synthesis and endoplasmic reticulum stress can be modulated by the hepatitis C virus envelope protein E2 through the eukaryotic initiation factor 2alpha kinasePERK. J Virol , 2003, 77(6): 3578–3585
15 Chan SW, Egan PA. Hepatitis C virus envelope proteins regulate CHOP via induction of the unfolded protein response. FASEB J , 2005, 19(11): 1510–1512
16 Wei YR, Wang D, Topczewski F, et al. Saturated fatty acids induce endoplasmic reticulum stress and apoptosis independently of ceramide in liver cells. Am J Physiol Endocrinol Metab , 2006, 291(2): E275-E281.
17 Pachikian BD, Neyrinck AM, Cani PD, et al. Hepatic steatosis in n-3 fatty acid depleted mice: focus on metabolic alterations related to tissue fatty acid composition. BMC Physiol, 2008, 8:21
18 Rutkowski DT, Wu J, Back SH, et al.UPR pathways combine to prevent hepatic steatosis caused by ER stress-mediated suppression of transcriptional master regulators. Dev Cell, 2008 , 15(6):829-840
19陈璐璐,李凝旭,邓向群.限食对高脂喂养大鼠肝脏内质网应激的影响.中国病理生理杂志, 2008, 24(3): 568–572
20 Ji C, Kaplowitz N. Betaine decreases hyperhomocysteinemia, endoplasmic reticulum stress, and liver injury in alcohol-fed mice. Gastroenterology, 2003, 124(5): 1488–1499
21 Esfandiari F, Villanueva JA, Wong DH, et al. Chronic ethanol feeding and folate deficiency activate hepatic endoplasmic reticulum stress pathway in micropigs. Am J Physiol Gastrointest Liver Physiol, 2005, 289(1): G54–G63
22 Perlmutter DH. The role of autophagy in alpha-1-antitrypsin deficiency: A specific cellular response in genetic diseases associated with aggregation-prone proteins. Autophagy, 2006, 2(4): 258–263
23 Jian BX , Hsieh CH, Chen J. Activation of endoplasmic reticulum stress response following trauma-hemorrhage . Biochim Biophys Acta, 2008, 1782(11) : 621–626
24 Nagy G, Kardon T, Wunderlich L, et al. Acetaminophen induces ER dependent signaling in mouse liver. Arch Biochem Biophys, 2007, 459(2): 273–279
25 Zhou H, Gurley E, Jarujaron S, et al. HIY protease inhibitors activate the unfolded protein response and disrupt lipid metabolism in primary hepatocytes. Am J Physiol, 2006, 291(6): G1071–G1080
26温韬,张海燕,卢静,等.内质网应激在四氯化碳致大鼠急性肝损伤中的作用探讨.胃肠病学和肝病学杂志, 2008, 17(10): 786-789
27姜山,谢青,周惠娟,等.Caspase-12活化在小鼠原代肝细胞凋亡中作用.肝脏, 2006, 11(1):12-14
28 Hiramatsu N, Kasai A, Du S, et al. Rapid, transient induction of ER stress in the liver and kidney after acute exposure to heavy metal: evidence from transgenic sensor mice. FEBS Lett, 2007, 581(10): 2055–2059