高糖对人肾小球系膜细胞SREBP-1、FAS表达的影响

目的探讨高糖环境中人肾小球系膜细胞（human mesangial cells,HMC）SREBP-1、FAS表达。方法体外培养HMC细胞,随机分为正常糖组、高糖组,免疫细胞化学、Western Blot和RT-PCR方法检测固醇调节元件结合蛋白1（sterol regulatory element binding protein-1,SREBP-1）和脂肪酸合酶（fatty acid synthase,FAS）表达。采用脂质体转染技术将特异性SREBP-1质粒引入细胞内并进行表达,进一步采用RT-PCR方法检测脂肪酸合酶FAS的表达。结果与正常糖组比较,高糖培养的人肾小球系膜细胞固醇调节元件结合蛋白1前体和成熟体以及FAS mRNA表达均升高,差异有统计学意义。质粒转染后HMC细胞经免疫组化和Western blot检测证实特异性质粒能够在细胞内高表达SREBP-1蛋白,进一步对FAS的检测证实了FAS mRNA表达升高。结论高糖可诱导人肾小球系膜细胞固醇调节元件结合蛋白1和FAS表达增强且SREBP-1和FAS之间存在有直接关系。     

过表达apoA—I对小鼠非酒精性脂肪性肝炎作用的研究

目的研究非酒精性脂肪性肝炎（NASH）,脂肪酸（FFA）及载脂蛋白A1（apoA-I）三者之间的作用及其对预防治疗。NASH的意义。方法通过胆碱一蛋氨酸缺乏（MCD）饲料喂养的小鼠NASH模型,注射含有人apoA-I基因的腺病毒载体或对照腺病毒载体一周后,检测小鼠肝脏指数,血清谷丙转氨酶、谷草转氨酶水平,对肝脏切片进行油红O染色,检测血清和肝组织中甘油三酯（TG）,FFA及胆固醇（Ch01）的含量。结果与对照组相比,过量表达apoA—I的小鼠肝脏病变减轻,脂质堆积减少,血清谷丙转氨酶、谷草转氨酶水平有所下降,肝组织中TG,FFA及Chol含量明显降低,相应的其血清TG,Chol水平升高。结论过量表达apoA-I可通过减少小鼠肝脏中过量脂质的堆积而对NASH起到缓解作用。     

衣霉素诱导大鼠心肌细胞内质网应激凋亡模型的构建

目的：通过衣霉素诱导内质网应激建立新生大鼠心肌细胞凋亡模型。方法：不同浓度、不同时间的衣霉素作用于原代培养乳鼠心肌细胞,通过MTT实验和流式细胞术测定心肌细胞的存活率和凋亡率,Western blot检测内质网应激蛋白GRP78,CHOP表达水平。结果：①与阴性对照组相比,衣霉素具有损伤心肌细胞的作用,并呈现剂量与时间依赖关系（P〈0.05,n=12）。②通过流式细胞术判断心肌细胞死亡的性质,当衣霉素浓度为100ng/ml,作用72h时,心肌细胞存活率和凋亡率分别为57.4±3.2%（n=12）,25.9±5.8%（n=3）。提示衣霉素损伤细胞的形式主要为凋亡性死亡。③内质网应激蛋白GRP78和CHOP表达于6h开始增加,24h达到峰值,随后呈下降趋势。结论：应用衣霉素成功诱导SD乳鼠心肌细胞内质网应激凋亡模型,衣霉素的最佳诱导浓度为100ng/ml,作用时间为72h。     

SRO9基因参与调控酿酒酵母内质网应激反应

【目的】研究酵母SRO9基因在内质网应激(Endoplasmic reticulum stress,ERS)中的作用。【方法】利用PCR介导的同源重组方法构建SRO9基因缺失菌株,检测其在内质网应激诱导剂衣霉素处理条件下的克隆形成能力;通过比色法检测细胞内的H2O2含量,超氧化物歧化酶SOD活性和细胞增殖能力;通过实时荧光定量PCR检测内质网应激靶基因和超氧化物歧化酶编码基因SOD1及SOD2的转录水平。【结果】相对于野生型酵母菌株,SRO9基因缺失酵母菌株对内质网应激诱导剂衣霉素的抗性增强,参与内质网应激反应的靶基因转录上调;细胞内H2O2含量下降,SOD1、SOD2转录水平降低,总SOD活性降低;对氧化剂CHP和VK3的抵抗性减弱,复制寿命明显缩短。【结论】SRO9基因缺失酵母细胞对内质网应激诱导剂衣霉素的抗性增强,原因可能是由于SRO9基因缺失激活了细胞的内质网应激反应。     

内质网膜复合亚基3调控内质网应激影响人畸胎瘤细胞的存活

内质网膜蛋白复合物（endoplasmic reticulum membrane complex,EMC）在跨膜蛋白质的生物发生和膜整合中发挥重要作用。内质网膜复合亚基3(endoplasmic reticulum membrane complex 3,EMC3)是EMC的重要组成部分,但其在生殖细胞中发挥的作用未见报道。本研究通过实时荧光定量PCR法检测18周龄小鼠睾丸、肺、脾、下丘脑组织中的EMC3 mRNA表达水平差异。结果显示,小鼠睾丸中EMC3 mRNA表达水平较高。体外培养人畸胎癌细胞NCCIT,通过不同浓度衣霉素诱导细胞产生内质网应激(endoplasmic reticulum stress,ERS),应用实时荧光定量PCR法、蛋白质印迹法检测其中EMC3、葡萄糖调节蛋白78(glucose regulatory protein,GRP78)、CCAAT-增强子结合蛋白同源蛋白(CCAAT-enhancer-binding protein homologous protein,CHOP)的mRNA及其蛋白质表达水平。结果显示,相对于对照组,EMC3、GRP78、CHOP的mRNA与蛋白质水平表达极显著升高(P< 0.01),表明衣霉素成功诱导了NCCIT细胞产生内质网应激,EMC3在衣霉素诱导的内质网应激的精原细胞中,mRNA与蛋白质水平表达升高。以NCCIT细胞cDNA为模板,利用PCR法扩增EMC3基因片段,并将其与pRK5-myc载体连接,构建pRK5-myc-EMC3重组质粒,经双酶切鉴定及DNA测序表明：pRK5-myc-EMC3重组质粒构建成功。将重组质粒和空载体分别转染至NCCIT细胞中进行表达,应用实时荧光定量PCR法与CCK8法检测细胞中GRP78、CHOP的mRNA转录水平以及细胞活力。结果显示,EMC3转染组的GRP78、CHOP的mRNA水平表达显著升高(P< 0.05),细胞活性极显著降低(P< 0.01),表明EMC3可以在NCCIT细胞中调控内质网应激并抑制细胞存活的发生。综上表明,过表达EMC3能够在精原细胞中调控内质网应激抑制细胞存活,EMC3可能在精原细胞的内质网应激中发挥重要作用。     

不同程度内质网应激对巨噬细胞自噬的影响

目的：采用ox-LDL诱导巨噬细胞泡沫化模型,通过不同剂量衣霉素诱导巨噬细胞不同程度内质网应激,观察对其自噬的影响。方法：不同剂量衣霉素作用于小鼠巨噬细胞系RAW264．7,通过TUNEL染色检测其凋亡率,Westernblot检测内质网应激蛋白GRP78,以及自噬标志蛋白P62的表达水平。结果：与ox-LDL组和大剂量衣霉素组相比,小剂量衣霉素组可以显著减少巨噬细胞的凋亡（P〈0．01）;与ox-LDL组相比,小剂量衣霉素组上调内质网应激蛋白GRP78表达的同时,自噬标志蛋白P62适度下降（P〈0．01）;大剂量衣霉素组更为显著地上调了内质网应激蛋白GRP78表达,但同时自噬标志蛋白P62也显著增加（P〈0．01）。结论：小剂量衣霉素引起一定程度的内质网应激,可以激活适度的自噬,从而减少巨噬细胞的凋亡,可能有助于降低动脉粥样硬化的程度。     

不同程度内质网应激对巨噬细胞自噬的影响*

摘要目的：采用ox-LDL诱导巨噬细胞泡沫化模型,通过不同剂量衣霉素诱导巨噬细胞不同程度内质网应激,观察对其自噬的影 响。方法：不同剂量衣霉素作用于小鼠巨噬细胞系RAW264.7,通过TUNEL染色检测其凋亡率,Western blot检测内质网应激蛋白 GRP78,以及自噬标志蛋白P62 的表达水平。结果：与ox-LDL组和大剂量衣霉素组相比,小剂量衣霉素组可以显著减少巨噬细胞 的凋亡（P<0.01）;与ox-LDL 组相比,小剂量衣霉素组上调内质网应激蛋白GRP78表达的同时,自噬标志蛋白P62 适度下降（P< 0.01）;大剂量衣霉素组更为显著地上调了内质网应激蛋白GRP78 表达,但同时自噬标志蛋白P62 也显著增加（P<0.01）。结论：小 剂量衣霉素引起一定程度的内质网应激,可以激活适度的自噬,从而减少巨噬细胞的凋亡,可能有助于降低动脉粥样硬化的程 度。     

肝细胞表面去唾液酸糖蛋白受体的流式细胞分析

建立肝细胞表面去唾液酸糖蛋白受体（ASGPR）的流式细胞分析方法（FCM）,对正常及损伤鼠肝细胞、肝癌细胞（BEL-7402）表面的ASGPR作同步比较分析.以异硫氰酸荧光素标记的新半乳糖白蛋白（FITC-NGA）为ASGPR的特异性配体,以培养的正常肝细胞（L-02）为靶细胞,建立肝细胞表面ASGPR的FCM.测定并计算正常及损伤鼠肝细胞,BEL-7402细胞与同一浓度的FITC-NGA同步反应后的平均荧光强度（MIF）值.FITC-NGA与L-02细胞表面ASGPR趋近饱和结合的浓度为0.4 mg/L,该浓度下正常及损伤鼠肝细胞,BEL-7402细胞的MIF值分别为228.7、5.81、1.13.该结合可以被至少50倍于FITC-NGA的NGA或10 mmol/L的EDTA完全抑制.FCM能够良好地揭示FITC-NGA同ASGPR之间的受配体结合特性.该方法证实BEL-7402细胞表面几乎没有ASGPR,损伤鼠肝细胞表面ASGPR的数量较正常鼠肝细胞显著减少.     

基于载体的RNA干扰介导入核受体hLRH-1表达稳定抑制肝癌细胞BEL-7402的基因表达谱分析

为建立-hLRH-1表达稳定抑制的细胞系,我们构建了hLRH-1基因靶向的稳定RNA干涉载体（pSineohLRH-1）并导入肝细胞癌细胞BEL-7402。通过半定量RT-PCR分析发现,携有pSineohLRH-1的BEL-7402细胞其hLRH-1基因mRNA的表达抑制率达60％。此外,微阵列法基因表达谱分析结果表明,与未干涉的对照细胞相比,包括一些肿瘤相关的基因如Gadd45β和PTEN在内的405个基因在hLRH-1基因表达稳定下调的BEL-7402细胞中呈明显的表达差异,意示hLRH-1具比已知更为广泛的生物学功能。尽管hLRH-1与这些差异表达基因的确切关系尚有待进一步的实验探究,我们的发现仍为hLRH-1在肿瘤发生发展中可能的作用机制的揭示提供了新的线索。     

Endoplasmic reticulum stress in adipose tissue augments lipolysis

The endoplasmic reticulum (ER) is an organelle important for protein synthesis and folding, lipid synthesis and Ca²⁺ homoeostasis. Consequently, ER stress or dysfunction affects numerous cellular processes and has been implicated as a contributing factor in several pathophysiological conditions. Tunicamycin induces ER stress in various cell types in vitro as well as in vivo. In mice, a hallmark of tunicamycin administration is the development of fatty livers within 24–48 hrs accompanied by hepatic ER stress. We hypothesized that tunicamycin would induce ER stress in adipose tissue that would lead to increased lipolysis and subsequently to fatty infiltration of the liver and hepatomegaly. Our results show that intraperitoneal administration of tunicamycin rapidly induced an ER stress response in adipose tissue that correlated with increased circulating free fatty acids (FFAs) and glycerol along with decreased adipose tissue mass and lipid droplet size. Furthermore, we found that in addition to fatty infiltration of the liver as well as hepatomegaly, lipid accumulation was also present in the heart, skeletal muscle and kidney. To corroborate our findings to a clinical setting, we examined adipose tissue from burned patients where increases in lipolysis and the development of fatty livers have been well documented. We found that burned patients displayed significant ER stress within adipose tissue and that ER stress augments lipolysis in cultured human adipocytes. Our results indicate a possible role for ER stress induced lipolysis in adipose tissue as an underlying mechanism contributing to increases in circulating FFAs and fatty infiltration into other organs.     

Enhanced cellular cholesterol efflux by naringenin is mediated through inhibiting endoplasmic reticulum stress - ATF6 activity in macrophages

Naringenin improves lipoprotein profile and protects against cardiovascular disease. ATF6 is an endoplasmic reticulum (ER) stress sensor with the same activation processes with sterol regulator SREBPs. Clinical data revealed that ATF6 expression was associated with plasma cholesterol level. Here, we investigated whether naringenin was involved in the regulation of cholesterol efflux and tested the role of ER stress-ATF6 in the naringenin function. Results showed that naringenin increased cholesterol efflux to both apoA-I and HDL and gene expressions in ABCA1, ABCG1 and LXRα in RAW264.7 macrophages. Naringenin inhibited the cleaved ATF6 nuclear translocation and its target GRP78 and XBP-1 expressions. Naringenin-induced cholesterol efflux was modulated by treatment with ER stress inhibitor 4-phenylbutyric acid, inducer tunicamycin and ATF6 overexpression in RAW264.7 and/or THP-1 cells, which suggested the naringenin functions were mediated through inhibiting ER stress-ATF6 pathway. Next, we found high-fat diet (HFD) supplemented with naringenin increased by >1.2-fold in cholesterol efflux capacity in primary peritoneal macrophage in apoE−/− mice compared to only HFD-fed mice. The increase was significantly reduced by tunicamycin treatment. Naringenin decreased GRP78, XBP-1 and nuclear ATF6 levels in peritoneal macrophage and aorta and reduced atherosclerotic lesion at aortic root, but reversed by tunicamycin. These confirmed participation of ER stress-ATF6 in naringenin efficacy. Finally, we found naringenin promoted AKT phosphorylation; PI3K inhibitor LY294002 treatment increased nuclear ATF6 and reduced naringenin-enhanced ABCA1 expression and cholesterol efflux. We concluded naringenin as a regulator for cholesterol efflux, and the regulation was mediated by ATF6 branch of ER stress and PI3K/AKT pathway.     

Experimental evidence for therapeutic potential of taurine in the treatment of nonalcoholic fatty liver disease

The incidence of obesity is now at epidemic proportions and has resulted in the emergence of nonalcoholic fatty liver disease (NAFLD) as a common metabolic disorder that can lead to liver injury and cirrhosis. Excess sucrose and long-chain saturated fatty acids in the diet may play a role in the development and progression of NAFLD. One factor linking sucrose and saturated fatty acids to liver damage is dysfunction of the endoplasmic reticulum (ER). Although there is currently no proven, effective therapy for NAFLD, the amino sulfonic acid taurine is protective against various metabolic disturbances, including alcohol-induced liver damage. The present study was undertaken to evaluate the therapeutic potential of taurine to serve as a preventative treatment for diet-induced NAFLD. We report that taurine significantly mitigated palmitate-mediated caspase-3 activity, cell death, ER stress, and oxidative stress in H4IIE liver cells and primary hepatocytes. In rats fed a high-sucrose diet, dietary taurine supplementation significantly reduced hepatic lipid accumulation, liver injury, inflammation, plasma triglycerides, and insulin levels. The high-sucrose diet resulted in an induction of multiple components of the unfolded protein response in the liver consistent with ER stress, which was ameliorated by taurine supplementation. Treatment of mice with the ER stress-inducing agent tunicamycin resulted in liver injury, unfolded protein response induction, and hepatic lipid accumulation that was significantly ameliorated by dietary supplementation with taurine. Our results indicate that dietary supplementation with taurine offers significant potential as a preventative treatment for NAFLD.     

Endoplasmic reticulum stress impairs cholesterol efflux and synthesis in hepatic cells

Metabolic disorders such as type 2 diabetes cause hepatic endoplasmic reticulum (ER) stress, which affects neutral lipid metabolism. However, the role of ER stress in cholesterol metabolism is incompletely understood. Here, we show that induction of acute ER stress in human hepatic HepG2 cells reduced ABCA1 expression and caused ABCA1 redistribution to tubular perinuclear compartments. Consequently, cholesterol efflux to apoA-I, a key step in nascent HDL formation, was diminished by 80%. Besides ABCA1, endogenous apoA-I expression was reduced upon ER stress induction, which contributed to reduced cholesterol efflux. Liver X receptor, a key regulator of ABCA1 in peripheral cells, was not involved in this process. Despite reduced cholesterol efflux, cellular cholesterol levels remained unchanged during ER stress. This was due to impaired de novo cholesterol synthesis by reduction of HMG-CoA reductase activity by 70%, although sterol response element-binding protein-2 activity was induced. In mice, ER stress induction led to a marked reduction of hepatic ABCA1 expression. However, HDL cholesterol levels were unaltered, presumably because of scavenger receptor class B, type I downregulation under ER stress. Taken together, our data suggest that ER stress in metabolic disorders reduces HDL biogenesis due to impaired hepatic ABCA1 function.     

Effects of different fatty acids on BRL3A rat liver cell damage

To evaluate the effects of fatty acids on endoplasmic reticulum (ER) stress, oxidative stress, and lipid damage. We treated BRL3A rat liver cells with, linoleic (LA), linolenic, oleic (OA), palmitic (PA), palmitoleic (POA), or stearic (SA) acid for 12 hr. The characteristics of cell lipid deposition, oxidative stress indexes, ER stress markers, nuclear factor κB p65 (NF-κB p65), lipid synthesis and transport regulators, and cholesterol metabolism regulators were analyzed. Endoplasmic chaperones like glucose-regulated protein 78, CCAAT-enhancer-binding protein, NF-κB p65, hydrogen peroxide, and malonaldehyde in PA- and SA-treated cells were significantly higher than in other treated cells. Deposition of fatty acids especially LA and POA were significantly increased than in other treated cells. De novo lipogenesis regulators sterol regulatory element-binding protein 1c, fatty acid synthase, and acetyl-coenzyme A carboxylase 1 (ACC1) expression were significantly increased in all fatty acid stimulation groups, and PA- and SA-treated cells showed lower p-ACC1 expression and higher scd1 expression than other fatty acid groups. Very low-density lipoprotein synthesis and apolipoprotein B100 expression in free fatty acids treated cells were significantly lower than control. PA, SA, OA, and POA had shown significantly increased cholesterol synthesis than other treated cells. PA and SA showed the lower synthesis of cytochrome P7A1 and total bile acids than other fatty acids treated cells. Excess of saturated fatty acids led to severe ER and oxidative stress. Excess unsaturated fatty acids led to increased lipid deposition in cultured hepatocytes. A balanced fatty acid intake is needed to maintain lipid homeostasis.     

Overexpression of calreticulin fails to abolish its induction by perturbation of normal ER function.

Along with other endoplasmic reticulum (ER) Ca2+-binding proteins, notably the glucose-response proteins grp78 and grp94, expression of calreticulin is induced in response to perturbation of normal ER function. It has yet to be clearly defined how this stress is signaled from the ER to the nucleus in mammalian cells, particularly with regard to its initiation. Using a GFP-calreticulin fusion protein, we have generated and selected stably transfected HeLa cells that overexpress calreticulin to investigate whether the protein might be involved in signaling its own induction. Basal levels of endogenous calreticulin mRNA and protein were unaffected in these cells, indicating that overexpression alone does not induce a stress response. ER stress induced calreticulin expression in response to either thapsigargin or tunicamycin was equivalent in these cells to that seen in control, nontransfected cells, leading us to conclude that calreticulin is unlikely be involved in its own induction. Levels of the mRNA encoding the fusion protein were also increased by tunicamycin, but not thapsigargin, suggesting that, in agreement with our previous observations, inhibition of N-linked glycosylation may increase the stability of calreticulin mRNA. This indicates that in mammalian cells, there is more than one signaling pathway for the ER stress response.     

ACSL3 and GSK-3β are essential for lipid upregulation induced by endoplasmic reticulum stress in liver cells

The endoplasmic reticulum (ER) is essential for lipid biosynthesis, and stress signals in this organelle are thought to alter lipid metabolism. Elucidating the mechanisms that underlie the dysregulation of lipid metabolism in hepatocytes may lead to novel therapeutic approaches for the treatment of lipid accumulation. We first tested the effects of several inhibitors on lipid dysregulation induced by tunicamycin, an ER stress inducer. Triacsin C, an inhibitor of long-chain acyl-CoA synthetase (ACSL) 1, 3, and 4, was the most potent among these inhibitors. We then analyzed the expression of the ACSL family during ER stress. The expression of ACSL3 was induced by ER stress in HuH-7 cells and in mice livers. ACSL3 shRNA, but not ACSL1 shRNA, inhibited the induction of lipid accumulation. GSK-3β inhibitors attenuated ACSL3 expression and the lipid accumulation induced by ER stress in HuH-7 cells. shRNA that target GSK-3β also inhibited the upregulation of ACSL3 and lipid accumulation in HuH-7 and HepG2 cells. The hepatitis B virus mutant large surface protein, which is known to induce ER stress, increased the lipid content of cells. Similarly, Triacsin C, and GSK-3β inhibitors abrogated the lipid dysregulation caused by the hepatitis B virus mutant large surface protein. Altogether, ACSL3 and GSK-3β represent novel therapeutic targets for lipid dysregulation by ER stress.