大鼠非酒精性脂肪性肝病与内质网应激关系的研究

目的 通过建立大鼠非酒精性脂肪性肝病(NAFLD)模型,探讨内质网应激(ERS)在NAFLD中的作用.方法 36只雄性SD大鼠分为对照1组(n=8)、对照2组(n=8)、模型1组(n=10)和模型2组(n=10).对照组大鼠给予普通饲料,模型组大鼠则喂饲高脂饲料.分别于第12周末处死对照1组和模型1组,第20周末处死对照2组和模型2组大鼠.测定血清丙氨酸转氨酶(ALT)、天冬氨酸转氮酶(AST)、γ-谷氨酰转移酶(GGT)、碱性磷酸酶(ALP)、总蛋白(TP)、三酰甘油(TG)、总胆固醇(TC)、低密度脂蛋白(LDL)及高密度脂蛋白(HDL)水平.H-E和Masson染色观察肝组织脂肪变、炎症和纤维化的变化.实时定量PCR测定肝组织内GRP78、CHOP及proeaspase-12 mRNA表达情况.Western印迹法检测肝组织内procaspase-12和caspase-12蛋白的变化.结果 第12周末.模型1组大鼠ALT、AST、ALP、TC及LDL水平均较对照1组显著增高(P<0.01),而HDL水平显著降低(P<0.01);第20周末模型2组大鼠TC和LDL水平较模型1组显著增加(P<0.05).与对照1组相比,模型1组肝组织的脂肪变和炎症程度均明显增加(P值均<0.01);模型2组肝组织的脂肪变、炎症程度和纤维化分期较对照2组均明显增加(P值均<0.01),炎症程度和纤维化分期较模型1组明显加重(P值分别<0.05和0.01).模型组大鼠肝组织GRP78、CHOP及procaspase-12 mRNA水平在第12周末及第20周末时与对照组比较差异均无统计学意义(P值均>0.05).模型组caspase-12及其前体蛋白水平与对照组相比也无显著变化(P值均>0.05).结论 成功建立高脂饮食大鼠NAFLD模型.研究中未发现ERS,提示此模型中ERS诱导的肝脏损伤可能未参与NAFLD的发病机制.     

Irisin attenuates intestinal injury,oxidative and endoplasmic reticulum stress in mice with L-arginine-induced acute pancreatitis

BACKGROUND Acute pancreatitis(AP) is often associated with intestinal injury,which in turn exaggerates the progression of AP.Our recent study has shown that a low level of serum irisin,a novel exercise-induced hormone,is associated with poor outcomes in patients with AP and irisin administration protects against experimental AP.However,the role of irisin in intestinal injury in AP has not been evaluated.AIM To investigate the effect of irisin administration on intestinal injury in experimental AP.METHODS AP was induced in male adult mice by two hourly intraperitoneal injections of Larginine.At 2 h after the last injection of L-arginine,irisin(50 or 250 μg/kg body weight) or 1 m L normal saline(vehicle) was administered through intraperitoneal injection.The animals were sacrificed at 72 h after the induction of AP.Intestinal injury,apoptosis,oxidative and endoplasmic reticulum(ER) stress were evaluated.RESULTS Administration of irisin significantly mitigated intestinal damage,reduced apoptosis,and attenuated oxidative and ER stress in AP mice.In addition,irisin treatment also effectively downregulated serum tumor necrosis factor-alpha and interleukin-6 levels and alleviated injury in the pancreas,liver and lung of AP mice.CONCLUSION Irisin-mediated multiple physiological events attenuate intestinal injury following an episode of AP.Irisin has a great potential to be further developed as an effective treatment for patients with AP.     

Curcumin attenuates cytoplasmic/endoplasmic reticulum stress,apoptosis and cholinergic dysfunction in diabetic rat hippocampus

Metabolic Brain Disease - Diabetes mellitus (DM) is associated with the increased risk of the central nervous system complications as cerebrovascular disease, impaired cognition, dementia and...     

Schistosoma japonicum attenuates dextran sodium sulfateinduced colitis in mice via reduction of endoplasmic reticulum stress

AIM To elucidate the impact of Schistosoma(S.) japonicum infection on inflammatory bowel disease by studying the effects of exposure to S. japonicum cercariae on dextran sodium sulfate(DSS)-induced colitis. METHODS Infection was percutaneously established with 20 ± 2 cercariae of S. japonicum, and colitis was induced by administration of 3% DSS at 4 wk post infection. Weight change, colon length, histological score(HS) and disease activity index(DAI) were evaluated. Inflammatory cytokines, such as IL-2, IL-10 and IFN-γ, were tested by a cytometric bead array and real-time quantitative polymerase chain reaction(RT-PCR). Protein and m RNA levels of IRE1α, IRE1β, GRP78, CHOP, P65, P-P65, P-IκBα and IκBα in colon tissues were examined by Western blot and RT-PCR, respectively. Terminal deoxynucleotidyl transferase-mediated d UTP nick-end labeling positive cells, cleaved-caspase 3 expression and Bcl2/Bax were investigated to assess the apoptosis in colon tissues.RESULTS Mice infected with S. japonicum cercariae were less susceptible to DSS. Mice infected with S. japonicum cercariae and treated with DSS showed decreased weight loss, longer colon, and lower HS and DAI compared with mice treated with DSS alone. A substantial decrease in Th1/Th2/Th17 response was observed after infection with S. japonicum. Endoplasmic reticulum(ER) stress and the nuclear factor-kappa B(NF-κB) pathway were reduced in mice infected with S. japonicum cercariae and treated with DSS, along with ameliorated celluar apoptosis, in contrast to mice treated with DSS alone. CONCLUSION Exposure to S. japonicum attenuated inflammatory response in a DSS-induced colitis model. In addition to the Th1/Th2/Th17 pathway and NF-κB pathway, ER stress was shown to be involved in mitigating inflammation and decreasing apoptosis. Thus, ER stress is a new aspect in elucidating the relationship between helminth infection and inflammatory bowel disease(IBD), which may offer new therapeutic methods for IBD.     

Signalling danger: endoplasmic reticulum stress and the unfolded protein response in pancreatic islet inflammation

Protein synthesis is increased by several-fold in stimulated pancreatic beta cells. Synthesis and folding of (pro)insulin takes place in the endoplasmic reticulum (ER), and beta cells trigger the unfolded protein response (UPR) to upgrade the functional capacity of the ER. Prolonged or excessive UPR activation contributes to beta cell dysfunction and death in type 2 diabetes, but there is another side of the UPR that may be of particular relevance for autoimmune type 1 diabetes, namely, the cross-talk between the UPR and innate immunity/inflammation. Recent evidence, discussed in this review, indicates that both saturated fats and inflammatory mediators such as cytokines trigger the UPR in pancreatic beta cells. The UPR potentiates activation of nuclear factor κB, a key regulator of inflammation. Two branches of the UPR, namely IRE1/XBP1s and PERK/ATF4/CHOP, mediate the UPR-induced sensitisation of pancreatic beta cells to the proinflammatory effects of cytokines. This can contribute to the upregulation of local inflammatory mechanisms and the aggravation of insulitis. The dialogue between the UPR and inflammation may provide an explanation for the parallel increase in the prevalence of childhood obesity and type 1 diabetes.     

Rosiglitazone attenuates age- and diet-associated nonalcoholic steatohepatitis in male low-density lipoprotein receptor knockout mice

Nonalcoholic fatty liver disease (NAFLD) is a common complication of obesity that can progress to nonalcoholic steatohepatitis (NASH), a serious liver pathology that can advance to cirrhosis. The mechanisms responsible for NAFLD progression to NASH remain unclear. Lack of a suitable animal model that faithfully recapitulates the pathophysiology of human NASH is a major obstacle in delineating mechanisms responsible for progression of NAFLD to NASH and, thus, development of better treatment strategies. We identified and characterized a novel mouse model, middle-aged male low-density lipoprotein receptor (LDLR)(-/-) mice fed a high-fat diet (HFD), which developed NASH associated with four of five metabolic syndrome (MS) components. In these mice, as observed in humans, liver steatosis and oxidative stress promoted NASH development. Aging exacerbated the HFD-induced NASH such that liver steatosis, inflammation, fibrosis, oxidative stress, and liver injury markers were greatly enhanced in middle-aged versus young LDLR(-/-) mice. Although expression of genes mediating fatty acid oxidation and antioxidant responses were up-regulated in young LDLR(-/-) mice fed HFD, they were drastically reduced in MS mice. However, similar to recent human trials, NASH was partially attenuated by an insulin-sensitizing peroxisome proliferator-activated receptor-gamma (PPARγ) ligand, rosiglitazone. In addition to expected improvements in MS, newly identified mechanisms of PPARγ ligand effects included stimulation of antioxidant gene expression and mitochondrial β-oxidation, and suppression of inflammation and fibrosis. LDLR-deficiency promoted NASH, because middle-aged C57BL/6 mice fed HFD did not develop severe inflammation and fibrosis, despite increased steatosis. Conclusion: MS mice represent an ideal model to investigate NASH in the context of MS, as commonly occurs in human disease, and NASH development can be substantially attenuated by PPARγ activation, which enhances β-oxidation.     

Bofutsushosan,a Japanese herbal (Kampo) medicine,attenuates progression of nonalcoholic steatohepatitis in mice

Background

Obesity-induced liver disease (nonalcoholic fatty liver disease, NAFLD) is now the commonest cause of chronic liver disease in affluent nations. There are presently no proven treatments for NAFLD or its more severe stage, nonalcoholic steatohepatitis (NASH). Bofutsushosan (BTS), a Japanese herbal (Kampo) medicine, long used as an anti-obesity medicine in Japan and other Asian countries, has been shown to reduce body weight and improve insulin resistance (IR) and hepatic steatosis. The precise mechanism of action of BTS, however, remains unclear. To evaluate the ability of BTS to prevent the development of NASH, and determine the mediators and pathways involved.

Methods

C57BL/6 mice were injected intra-peritoneally with gold-thioglucose and fed a high-fat diet (HF) or HF diet admixed with either 2 or 5 % BTS for 12 weeks. The effectiveness of BTS in attenuating features of NASH and the mechanisms through which BTS attenuated NASH were then assayed through an assessment of the anthropometric, radiological, biochemical and histological parameters.

Results

BTS attenuated the progression of NASH through induction of adiponectin and its receptors along with an induction of PPAR-α and PPAR-γ, decreased expression of SREBP-1c, increased hepatic fatty acid oxidation and increased hepatic export of triglycerides. BTS moreover, reduced IR through phosphorylation of the protein kinase, Akt.

Conclusions

BTS through induction of adiponectin signaling and Akt attenuated development of NASH. Identification of the active entity in BTS should allow development of novel treatments for NASH.     

血管钠肽抑制内质网应激减轻糖尿病大鼠缺血/再灌注心肌损伤

目的 观察人工合成的钠尿肽——血管钠肽（VNP）对糖尿病（DM）大鼠心肌缺血/再灌注（MI/R）损伤的影响及机制。方法 高脂饲料喂养SD大鼠4周后,注射链脲霉素STZ（25 mg/kg,i.p.）,1周后随机血糖≥11.1 mmol/L为DM模型构建成功,常规制备MI/R（30 min/4 h）模型。将大鼠随机分为4组:假手术组、MI/R组、DM+假手术组、DM+MI/R组。多导生理记录仪检测左室压上升/下降最大速率（±LVdP/dtmax）,Evans blue-TTC双染色法检测心肌梗死面积,TUNEL法进行心肌细胞凋亡测试、试剂盒检测caspase-3活性,Western blot检测GRP78、Chop和PKG等蛋白表达。结果 与对照组相比,DM大鼠MI/R心肌损伤加重。VNP治疗（再灌前10 min,给予100 μg/kg,i.v）可显著减轻DM大鼠MI/R损伤,包括增强±LVdP/dtmax,降低心梗范围、死亡率与Caspase-3活性（n=8,P<0.05）。此外,VNP可降低内质网应激相关蛋白GRP78、CHOP表达（n=3,P<0.01）。VNP上述效应可同时被PKG阻断剂KT-5823（再灌前20 min,给予0.5 mg/kg,i.p）抑制、并被cGMP衍生物8-Br-cGMP（1 mg/kg）模拟（P<0.05,P<0.01）。用内质网应激抑制剂TUDCA（50 mg/kg）预处理DM大鼠,并不能增强VNP的心肌保护作用。结论 VNP治疗可减轻DM性MI/R损伤,其机制可能与通过cGMP-PKG信号抑制内质网应激有关,提示VNP对DM性缺血性心脏病具有潜在治疗价值。     

Betaine decreases hyperhomocysteinemia,endoplasmic reticulum stress,and liver injury in alcohol-fed mice

BACKGROUND & AIMS: Alcohol-induced hyperhomocysteinemia has been reported in rats and humans. Hyperhomocysteinemia has been associated with endoplasmic reticulum (ER) stress leading to the activation of ER-dependent apoptosis or up-regulation of lipid synthesis. This novel ER stress mechanism of alcoholic liver injury was studied in the model of intragastric alcohol-fed mice. METHODS: Effects of alcohol on gene expression were analyzed using cDNA microarrays, RT-PCR, and Western blots over a period of 6 weeks. Liver injury was examined by histologic staining and TUNEL. RESULTS: We observed fatty liver, increased hepatic necroinflammation and apoptosis, and hyperhomocysteinemia. Of 1176 toxicology-related genes, glucose-regulated proteins (GRP-78 and -94), growth arrest/DNA damage-inducible protein 153 (CHOP/GADD153), and caspase-12 indicative of an ER stress response were among the alcohol-responsive genes. Sterol regulatory element binding protein (SREBP-1) and HMG-CoA reductase also were enhanced with alcohol administration. RT-PCR and selective Western blots confirmed the alcohol-induced expression of ER stress-related apoptosis and lipid synthesis genes. Addition of 0.5% and maximal 1.5% betaine to the alcohol diet reduced the elevated level of plasma homocysteine by 54% and more than 80% accompanied by a decrease in hepatic lipids and ER stress response. Betaine did not attenuate the ethanol-induced increase in tumor necrosis factor alpha or CD14 mRNA. CONCLUSIONS: The results strongly suggest that alcohol may modulate both apoptotic and fat synthetic gene expression through homocysteine-induced ER stress in chronic alcoholic mouse liver and that correction of hyperhomocysteinemia by betaine or other approaches may be useful to prevent alcoholic liver disease.     

微生物源性抗氧化剂对diquat诱导的小鼠肝脏氧化应激、内质网应激和功能的影响

目的以diquat诱导的小鼠氧化应激为模型,研究微生物源性抗氧化剂(MA)对小鼠肝脏氧化应激、内质网应激、细胞凋亡和功能的影响。方法选择体质量16~18 g的C57BL/6雌性小鼠18只,随机分为3组,每组6只。抗氧化剂组小鼠灌胃MA,对照组和模型组小鼠灌胃等量等渗盐水,饲养22 d后,模型组和抗氧化剂组腹腔注射diquat溶液,对照组小鼠注射等量等渗盐水,处理3h后处死小鼠采集肝组织。肝脏组织制成10%匀浆后,依据试剂盒说明书检测自由基含量、丙二醛(MDA)含量、抗氧化酶活性、天冬氨酸转氨酶(AST)和丙氨酸转氨酶(ALT)活性,用qRT-PCR检测内质网应激和细胞凋亡相关基因表达情况。肝脏组织制成10%匀浆后,依据试剂盒说明书检测自由基含量、丙二醛(MDA)含量、抗氧化酶活性、天冬氨酸转氨酶(AST)和丙氨酸转氨酶(ALT)活性,用qRT-PCR检测内质网应激和细胞凋亡相关基因表达情况。多组间数据比较采用单因素方差分析。结果过氧化氢在对照组、模型组、抗氧化剂组分别为(8.74±1.38)、(11.44±1.01)、(9.81±0.98)mmol/g,组间比较差异有统计学意义(F=7.640,P<0.05)。对照组、模型组和抗氧化剂组MDA的含量分别为(0.65±0.07)、(0.86±0.18)、(0.70±0.05)nmol/mg,组间比较差异有统计学意义(F=5.406,P<0.05)。模型组AST活性为(146.68±4.29)U/g,显著高于对照组的(125.64±15.69)U/g与抗氧化剂组的(126.57±1.82)U/g,F=6.192,P<0.05。实时定量PCR结果显示,与对照组相比,模型组蛋白激酶R样内质网激酶(PERK)和活化转录因子6(ATF6)基因相对表达量显著升高,分别为1.880±0.442和1.800±0.380(F值分别为7.702、10.815,P值均<0.05);与模型组相比,抗氧化剂组PERK和ATF6基因相对表达量显著降低,分别为1.310±0.333、1.180±0.204(P值均<0.05)。细胞凋亡相关基因表达结果显示,抗氧化剂组caspase3和caspase8基因相对表达量分别为1.136±0.381、1.593±0.407,与模型组的1.572±0.127和2.843±0.973相比,显著降低(F值分别为12.800、7.657,P值均<0.05)。结论微生物源性抗氧化剂减弱diquat诱导的小鼠肝脏氧化应激、内质网应激和肝细胞凋亡,改善肝脏功能。     

杞蓟制剂对非酒精性脂肪性肝炎大鼠肝组织核转录因子-kB／IkB的影响

目的 观察非酒精性脂肪性肝炎（NASH）大鼠肝组织P65（NF-kB亚型）、核转录因子-kB抑制因子（IkB）-α（IkB亚型）蛋白和mRNA表达情况及杞蓟制剂对其影响。方法 采用高脂饮食复制大鼠NASH模型。实验分正常组对照、模型组、杞蓟制剂组、复方蛋氨酸胆碱片组。测定肝组织P65、IkB—α蛋白和mRNA的表达。结果与正常组相比，模型组大鼠肝组织P65、IkB—蛋白和mRNA表达明显增强（P〈0．01），且P65蛋白主要在胞核分布；与模型组相比，杞蓟制剂组大鼠肝组织P65、IkB—α蛋白和mRNA的表达明显降低（P〈0．05）。复方蛋氨酸胆碱片组大鼠肝组织P65、IkB-α蛋白和mRNA的表达虽有所降低，但与模型组相比差异无统计学意义（P〉0．05）。结论 NF—kB蛋白和mRNA表达增强及NF-kB蛋白活化增强参与了NASH的发病。杞蓟制剂能够抑制NF-kB（P65）蛋白和mRNA的表达，从而能防治NASH。     

Glucose-induced beta cell dysfunction in vivo in rats: link between oxidative stress and endoplasmic reticulum stress

Aims/hypothesis

Endoplasmic reticulum (ER) stress has been implicated in glucose-induced beta cell dysfunction. However, its causal role has not been established in vivo. Our objective was to determine the causal role of ER stress and its link to oxidative stress in glucose-induced beta cell dysfunction in vivo.

Methods

Healthy Wistar rats were infused i.v. with glucose for 48?h to achieve 20?mmol/l hyperglycaemia with or without the co-infusion of the superoxide dismutase mimetic tempol (TPO), or the chemical chaperones 4-phenylbutyrate (PBA) or tauroursodeoxycholic acid (TUDCA). This was followed by assessment of beta cell function and measurement of ER stress markers and superoxide in islets.

Results

Glucose infusion for 48?h increased mitochondrial superoxide and ER stress markers and impaired beta cell function. Co-infusion of TPO, which we previously found to reduce mitochondrial superoxide and prevent glucose-induced beta cell dysfunction, reduced ER stress markers. Similar to findings with TPO, co-infusion of PBA, which decreases mitochondrial superoxide, prevented glucose-induced beta cell dysfunction in isolated islets. TUDCA was also effective. Also similar to findings with TPO, PBA prevented beta cell dysfunction during hyperglycaemic clamps in vivo and after hyperglycaemia (15?mmol/l) for 96?h.

Conclusions/interpretation

Here, we causally implicate ER stress in hyperglycaemia-induced beta cell dysfunction in vivo. We show that: (1) there is a positive feedback cycle between oxidative stress and ER stress in glucose-induced beta cell dysfunction, which involves mitochondrial superoxide; and (2) this cycle can be interrupted by superoxide dismutase mimetics as well as chemical chaperones, which are of potential interest to preserve beta cell function in type 2 diabetes.