Pre‐ischemia melatonin treatment alleviated acute neuronal injury after ischemic stroke by inhibiting endoplasmic reticulum stress‐dependent autophagy via PERK and IRE1 signalings

Melatonin has demonstrated a potential protective effect in central nervous system. Thus, it is interesting to determine whether pre‐ischemia melatonin administration could protect against cerebral ischemia/reperfusion (IR)‐related injury and the underlying molecular mechanisms. In this study, we revealed that IR injury significantly activated endoplasmic reticulum (ER) stress and autophagy in a middle cerebral artery occlusion mouse model. Pre‐ischemia melatonin treatment was able to attenuate IR‐induced ER stress and autophagy. In addition, with tandem RFP‐GFP‐LC3 adeno‐associated virus, we demonstrated pre‐ischemic melatonin significantly alleviated IR‐induced autophagic flux. Furthermore, we showed that IR induced neuronal apoptosis through ER stress related signalings. Moreover, IR‐induced autophagy was significantly blocked by ER stress inhibitor (4‐PBA), as well as ER‐related signaling inhibitors (PERK inhibitor, GSK; IRE1 inhibitor, 3,5‐dibromosalicylaldehyde). Finally, we revealed that melatonin significantly alleviated cerebral infarction, brain edema, neuronal apoptosis, and neurological deficiency, which were remarkably abolished by tunicamycin (ER stress activator) and rapamycin (autophagy activator), respectively. In summary, our study provides strong evidence that pre‐ischemia melatonin administration significantly protects against cerebral IR injury through inhibiting ER stress‐dependent autophagy. Our findings shed light on the novel preventive and therapeutic strategy of daily administration of melatonin, especially among the population with high risk of cerebral ischemic stroke.     

Caspase-12在D-氨基半乳糖联合脂多糖诱导小鼠急性肝功能衰竭中的表达及作用

目的探讨Caspase-12在D-氨基半乳糖(D-Gal)/脂多糖(LPS)诱导小鼠急性肝功能衰竭发生发展过程中表达水平的变化及Caspase-12介导的内质网应激肝细胞凋亡途径在急性肝功能衰竭中的作用。方法以D-Gal/LPS联合腹腔注射诱导小鼠急性肝功能衰竭建立实验模型,在不同时间点动态检测血清氨基转移酶水平和观察肝组织病理变化,评估肝细胞凋亡和肝坏死演变过程;应用琼脂糖凝胶电泳检测肝细胞DNA凋亡条带;用半定量逆转录聚合酶链反应检测肝组织中Caspase-12 mRNA表达水平;west- ern blot检测Caspase-12、Bip/GRP78蛋白表达。结果药物诱导5h时肝组织中典型凋亡细胞增多,血清丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)开始升高,Caspase-12 mRNA表达明显增加, Caspase-12蛋白量表达反而减少;7h镜下出现大量肝细胞凋亡和坏死,ALT、AST水平达高峰,分别为(2564±1384)U/L和(1198±497)U/L,正常对照组为(59±17)U/L和(135±12)U/L,Caspase- 12 mRNA表达仍增加,Caspase-12蛋白表达量继续降低;9h 肝细胞坏死明显,伴散在凋亡细胞,ALT、AST水平明显下降,Caspase-12 mRNA表达较7 h下降。Bip/GRP78蛋自表达从5 h开始,至7 h逐渐增加。结论D-Gal/LPS诱导小鼠急性肝功能衰竭早期Caspase-12 mRNA表达水平逐渐升高,后期(7-9 h)降低,与肝细胞凋亡发生的时相一致;Caspase-12蛋白酶因内质网应激而被大量活化,提示Caspase-12介导的内质网应激肝细胞凋亡参与炎症性急性肝功能衰竭的发生发展,是急性肝功能衰竭中肝细胞损伤的重要机制之一,提示早期干预Caspase-12表达及活化对急性肝功能衰竭可能具有保护作用。     

异丙肾上腺素对心肌细胞内钙释放和肌浆网内钙容量的影响

目的 :心肌细胞膜上的 β-肾上腺素受体的激活对兴奋 -收缩耦联 （ECC）过程有重要的调节作用。本课题利用异丙肾上腺素 （ISO,1μmol/ L）激活 β-肾上腺素受体从而研究其对源自心肌细胞肌浆网的胞内钙释放 （ECC的重要环节 ）和肌浆网内钙容量的影响 ,进而分析钙释放与钙容量之间的关系。方法 :局部场刺激作用于成年大鼠心肌细胞 ,促使后者产生动作电位 ,进而诱发胞内钙瞬变 （ACT） ,由 ACT可估测胞内钙释放。肌浆网内钙容量则由咖啡因 （2 0 m mol/ L）诱发的钙瞬变 （CCT）估测。实验结果均由 Zeiss L SM- 5 10激光共聚焦显微镜系统记录。结果 :ISO作用下的 ACT峰值为 10 .2 9± 0 .35 （n=13）比正常情况下的 5 .74± 0 .2 7（n=18）高 （P<0 .0 1）。 ISO作用下的CCT峰值为 11.2 3± 0 .2 9（n=13）比正常情况下的 7.6 2± 0 .2 4 （n=18）高 （P<0 .0 1）。结论 :ISO可明显地提高心肌细胞内钙释放量和肌浆网内的钙容量。不管有无 ISO存在 ,胞内钙释放量总是只占肌浆网内钙容量的一部分。在正常情况下 ,心肌的钙释放量有较大的储备能力 ,且此储备可因β-肾上腺素受体的激活而动员。     

Seipin mutation at glycosylation sites activates autophagy in transfected cells via abnormal large lipid droplets generation

Aim:

Seipin is a protein that resides in endoplasmic reticulum, and involved in both lipid metabolic disorders and motor neuropathy. The aim of this study was to investigate the effects of mutant seipin on autophagy system and the morphology of lipid droplets in vitro.

Methods:

HEK-293, H1299 and MES23.5 cells were transfected with the plasmids of mutated seipin at glycosylation sites (N88S or S90L) and GFP-LC3 plasmids. The cells were subjected to immunofluorescence and flow cytometry assays, and the cell lysates were subjected to immunoblot analysis. Nile Red was used to stain the lipid droplets in the cells.

Results:

Overexpression of the mutated seipin proteins N88S or S90L activated autophagy in the 3 cell lines, and substantially altered the sub-cellular distribution of the autophagosome marker GFP-LC3, leading to a number of large vacuoles appearing in the cytoplasm. The sub-cellular location of GFP-LC3 and mutated seipin proteins highly overlapped. Moreover, and the mutated seipin proteins caused diffuse small lipid droplets to fuse into larger lipid droplets. Treatment of mutated seipin-transfected cells with the autophagy inhibitor 3-MA (5 mmol/L) facilitated the fusion of mutated seipin-induced large vacuoles. The protein glycosylation inhibitor tunicamycin could mimic the mutated seipin-induced effects, and treatment of the wild-type seipin-transfected cells with tunicamycin (2.5 μg/mL) produced similar morphological and biochemical properties as in the mutated seipin-transfected cells.

Conclusion:

The mutation of seipin at glycosylation sites disrupt its function in regulating lipid droplet metabolism, and the autophagy acts as an adaptive response to break down abnormal lipid droplets. The interruption of autophagy would accelerate the fusion of abnormal lipid droplets.     

骨桥蛋白通过内质网应激诱导C2C12肌细胞胰岛素抵抗

目的:探讨骨桥蛋白(osteopontin,OPN)对C2C12肌细胞胰岛素抵抗的影响及其可能的机制。方法:低血清培养辅以胰岛素处理诱导C2C12成肌细胞分化为C2C12肌细胞,蛋白免疫印迹检测蛋白质的表达,离心法分离细胞膜,葡萄糖摄取试剂盒定量葡萄糖摄取。结果:(1)骨桥蛋白以剂量依赖和时间依赖方式抑制胰岛素刺激的蛋白激酶B(Akt)的磷酸化,并抑制胰岛素所致的葡萄糖转运体4(Glut4)膜位移和葡萄糖的摄取;而特异性的抗OPN受体CD44抗体预处理可逆转上述变化。(2)OPN可诱导C2C12肌细胞的内质网应激,并促进c-Jun氨基端激酶(JNK)的磷酸化。(3)内质网应激抑制剂4-苯基丁酸(4-PBA)可降低OPN所增加的JNK磷酸化,恢复胰岛素刺激所致的Glut4的膜位移以及葡萄糖摄取。结论:骨桥蛋白通过诱导C2C12肌细胞内质网应激导致胰岛素抵抗。本研究为揭示骨桥蛋白在胰岛素抵抗和糖尿病中的作用提供了新的实验依据和理论解释。     

糖尿病大鼠视网膜中内质网应激蛋白、HIF-1α和血管内皮生长因子的表达

目的：探索糖尿病大鼠视网膜中内质网应激相关BIP (蛋白重链结合蛋白)、HIF-1α(低氧诱导因子-1α)和VEGF (血管内皮生长因子)的表达及意义。
　　方法：72只雄性SD大鼠,随机分为6组：正常2月对照组(C2m),糖尿病2月组(D2m),正常4月对照组(C4m),糖尿病4月组(D4m),正常6月对照组(C6m),糖尿病6月组(D6m),每组各12只。实验组给予一次性腹腔注射65mg/kg STZ建立糖尿病模型。 ELISA法检测BIP、HIF-1α和VEGF表达水平,免疫组化法观察大鼠BIP、HIF-1α和VEGF视网膜内定位情况。
　　结果：BIP表达随DM 病程的延长而增加( P<0.05),且DM各组与对照组相比,其差异均有统计学意义( P<0.01)。 HIF-1α在DM组中表达较对照组增加,其差异有统计学意义(P<0.05),但DM组各组间差异无统计学意义。 VEGF蛋白在D2 m组与对照组间差别无统计学意义,但 D4 m、D6 m 与对照组间差别有统计学意义( P<0.05),且D4 m和D6 m两组间差别有统计学意义。免疫组化：BIP在对照组视网膜神经节细胞层中少量表达,DM组主要分布于视网膜内核层和神经节细胞层;HIF-1α在对照组基本不表达, DM组各层均有表达;VEGF在对照组大鼠中视网膜各层中少量表达,而DM组大鼠的内核层、外核层及视网膜血管、神经节细胞层中 VEGF 阳性表达。
　　结论：糖尿病大鼠视网膜组织中的 BIP、HIF-1α、VEGF均较对照组增加且随糖尿病病程进展而增加,内质网应激和低氧诱导因子途径均可能在糖尿病视网膜病变的进展中起重要作用。     

内质网应激在肝脏疾病中的作用研究进展

内质网是细胞内蛋白质、脂类和糖类的重要合成基地,是细胞内钙离子的储存场所,与物质运输、物质交换、解毒作用密切相关。内质网应激是细胞的一种重要自我防御机制,能提高细胞对环境变化的适应能力,但过强过久的内质网应激则会引起不可逆的细胞损伤甚至凋亡。研究发现,内质网应激与多器官多种疾病相关。肝细胞中含有大量的内质网,对内质网应激更为敏感,许多肝脏疾病如肝细胞癌、药物性肝损伤、非酒精性脂肪性肝病、肝胰岛素抵抗等的发病机制均与内质网应激有关。本文就内质网应激对各种肝脏疾病的影响作一概述。     

Endoplasmic stress-inducing variants in carboxyl ester lipase and pancreatic cancer risk

BackgroundEndoplasmic reticulum (ER) stress-inducing variants in several pancreatic secretory enzymes have been associated with pancreatic disease. Multiple variants in CEL, encoding carboxyl ester lipase, are known to cause maturity-onset diabetes of the young (MODY8) but have not been implicated in pancreatic cancer risk.MethodsThe prevalence of ER stress-inducing variants in the CEL gene was compared among pancreatic cancer cases vs. controls. Variants were identified by next-generation sequencing and confirmed by Sanger sequencing. Variants of uncertain significance (VUS) were assessed for their effect on the secretion of CEL protein and variants with reduced protein secretion were evaluated to determine if they induced endoplasmic reticulum stress.ResultsER stress-inducing CEL variants were found in 34 of 986 cases with sporadic pancreatic ductal adenocarcinoma, and 21 of 1045 controls (P = 0.055). Most of the variants were either the CEL-HYB1 variant, the I488T variant, or the combined CEL-HYB1/I488T variant; one case had a MODY8 variant.ConclusionThis case/control analysis finds ER stress-inducing CEL variants are not associated with an increased likelihood of having pancreatic cancer.