Effect of hypoxia and glutamine or glucose deprivation on the expression of retinoblastoma and retinoblastoma-related genes in ERN1 knockdown glioma U87 cell line

The expression of retinoblastoma and several retinoblastoma-related genes was studied in glioma cell line U87 and its subline with knockdown of ERN1 (endoplasmic reticulum—nuclei-1), the main endoplasmic reticulum stress sensing and signaling enzyme. It was shown that a blockade of the ERN1 enzyme function increases the expression levels of retinoblastoma, retinoblastoma-like 1 and most retinoblastoma related genes: EID1, JARID1B, E2F1, E2F3, RBAP48 and CTIP, does not change RNF40 and RBAP46 and decreases KDM5A. We have also demonstrated that hypoxia reduces the expression levels of retinoblastoma, EID1, and E2F1 in ERN1-deficient glioma cells only. At the same time, the expression levels of retinoblastoma-like 1, E2F3, RBAP46, RBAP48 and CTIP decrease, while JARID1B and RBBP2 increase in both types of cells in hypoxic conditions, but the expression is much stronger in cells with suppressed function of ERN1. The expression level of JARID1B and KDM-5A mRNA is also enhanced in glutamine deprivation condition in both tested cell types, moreover, this effect is amplified by the blockade of the ERN1 enzyme function. The expression levels of retinoblastoma, EID1, RBAP48, and E2F3 are decreased in glutamine deprivation condition only in ERN1-deficient glioma cells, but RBL1, CTIP, RBAP46, and E2F1—in both tested cell types with more significant effect in ERN1-deficient cells. Glucose deprivation condition leads to a decrease of expression levels of retinoblastoma, RBL1, E2F3, RBAP46, and RBAP48 in both used cell types and of EID1 and E2F1 only in glioma cells with suppressed function of signaling enzyme ERN1. Thus, expression levels of retinoblastoma and most retinoblastoma-related genes are increased under a blockade of ERN1 enzyme function and significantly changed in hypoxia, glucose or glutamine deprivation conditions both in control U87 cells and ERN1-deficient cells, but inhibition of the unfolded protein response sensor ERN1 predominantly enhances these effects. Moreover, it is possible that the induction of the expression of retinoblastoma and most retinoblastoma-related genes after knockdown of ERN1 plays an important role in suppression of glioma proliferation.

雷公藤对糖尿病大鼠肾皮质GLUT-1 mRNA表达的影响及其意义

目的探讨雷公藤对糖尿病(DM)大鼠肾脏GLUT-1mRNA表达的影响及其意义。方法采用链脲佐菌素(STZ)腹腔注射法建立DM动物模型。将46只3月龄160～190g大鼠(雌雄各半)随机分为正常对照组,DM模型组,雷公藤组(6.7mg·kg-1.d-1)。第4、8、12周末各组处死4只大鼠并收集标本,记录体重、右肾重、检测血糖、血尿素氮、肌酐、24h尿蛋白定量。用RT-PCR方法检测肾皮质GLUT-1mRNA表达水平。肾组织行HE、PAS染色。结果①与正常对照组相比,DM模型组大鼠造模成功后血糖明显增高(P<0.01),但24h尿蛋白排泄量无明显增高(P>0.05);4w时体重、肾重指数、尿素氮、肌酐、24h尿蛋白排泄量、肾皮质GLUT-1mRNA表达均明显增加(P均<0.01),12w时增加更加显著。与DM模型组相比,雷公藤组8w时血糖、尿素氮、肌酐降低(P均<0.05),24h尿蛋白排泄量和肾皮质GLUT-1mRNA表达量均明显下降(P均<0.01);12w时体重增加(P<0.05),肾重指数才明显下降(P<0.01)。②肾组织HE、PAS染色病理学观察:DM模型组光镜下肾小球肥大,系膜细胞增生,系膜基质弥漫性或结节性增多,肾小球、肾小管基底膜增厚,而雷公藤组这些病理改变明显减轻。结论雷公藤能明显减少DM大鼠尿蛋白排泄量,抑制肾脏肥大、减轻肾脏的纤维化硬化程度。其机制可能是下调GLUT-1mRNA的表达量及功能活性,最终延缓DN的发展。

断奶仔猪小肠钠葡萄糖转运蛋白1和葡萄糖转运蛋白2 mRNA表达变化及饲粮添加谷氨酰胺对其的影响

本试验旨在研究仔猪断奶后小肠黏膜钠葡萄糖转运蛋白1（SGLT1）和葡萄糖转运蛋白2（GLUT2）mRNA表达的发育性变化规律及谷氨酰胺是否对SGLT1和GLuT2mRNA的表达产生影响。选择21日龄断奶的杜×长×大仔猪69头，断奶当天屠宰3头猪，其余66头随机分成2组，每组3个重复，每个重复11头仔猪。对照组饲喂基础饲粮（不添加谷氨酰胺），试验组饲喂基础饲粮＋1％谷氨酰胺。断奶后第3、5、7、14天每组分别屠宰3头。取十二指肠、空肠和回肠黏膜样品，通过荧光定量RT—PCR方法测定SGLT1和GLUT2 mRNA的表达量。结果表明：1）SGLT1和GLUT2 mRNA的表达量在不同肠段之间无显著差异（P〉0．05）。2）十二指肠、空肠中SGLT1 mRNA表达量在断奶后先降低，断奶后第3天开始升高，第14天达峰值，回肠SGLT1 mRNA表达量在断奶后先升高，断奶后第7天较低，第14天为峰值；GLUT2 mRNA在十二指肠和空肠中的表达量从断奶后第3～7天呈现持续上升的趋势，第7天达峰值，但在第14天降至断奶当天水平，回肠GLUT2 mRNA表达量在断奶后逐渐升高，第5天达峰值，而后逐渐回落至断奶当天水平。3）饲粮添加1％谷氨酰胺对SGLT1和GLUT2 mRNA表达量无显著影响。结果提示：断奶仔猪十二指肠、空肠和回肠SGLT1和GLUT2 mRNA表达量无显著差异，但均随断奶后的时间而变化，谷氨酰胺对SGLT1和GLUT2在转录水平上无显著影响。

Expression of casein kinase genes in glioma cell line U87: Effect of hypoxia and glucose or glutamine deprivation

The endoplasmic reticulum-nuclei-1 (ERN1) sensing and signaling enzyme mediates a set of complex intracellular signaling events known as the unfolded protein response. We have studied the effect of hypoxia and ischemic conditions (glucose or glutamine deprivation) on the expression of several casein kinase-1 and -2 genes in glioma U87 cells and its subline with suppressed function of ERN1. It was shown that blockade of ERN1, the key endoplasmic reticulum stress sensor, leads to an increase in the expression levels of casein kinase-1G2, -1E, -2B and NUCKS1 mRNA, but suppresses casein kinase-1A1, -1D and -2A1. Moreover, the expression levels of casein kinase-1A1, -1D and 1G3 as well as casein kinase-2A1 and -2A2 mRNAs are significantly increased under glutamine dep- rivation conditions both in control and ERN1- deficient glioma cells. At the same time, casein kinase-1E, -2B and NUCKS1 mRNA expression levels are also increased under this condition, but only in cells with suppressed function of ERN1. The expression level of NUCKS1 mRNA, however, is decreased both in control glioma cells and in genetically modified cells, but casein kinase-1G2—only in control U87 cells. Cell exposure to glucose deprivation conditions enhances the expression levels of casein kinase- 1D, 1G3, -1E and -2A1 in both types of glioma cells used, but casein kinase-2B expression levels increase only in cells with suppressed function of ERN1. Hypoxia induces or suppresses the expression of most of the studied genes mainly in ERN1-knockdown cells only. Results of this study show that hypoxia as well as glutamine and glucose deprivation conditions change the expression level most of casein kinase genes and that these effects are dependent on ERN1 signaling enzyme function.

益气养阴方对早期糖尿病肾病大鼠肾功能及MCP-1 mRNA表达的影响

目的:观察益气养阴方对早期糖尿病肾病大鼠的疗效并探讨其作用机制。方法:48只Wistar大鼠随机分为正常组、模型组、益气养阴方组、西药组。除正常组外,其余组均采用单侧肾脏切除、高脂高糖饮食联合STZ制备糖尿病肾病大鼠模型,观察各组大鼠治疗后血糖、尿白蛋白、血肌酐、尿素氮、单核细胞趋化蛋白-1(MCP-1)mRNA的变化。结果:模型组大鼠血糖、尿白蛋白较正常组高(P<0.05),益气养阴方组和西药组大鼠各项指标均好转,其中益气养阴方组血糖值及西药组血糖值、尿白蛋白值与模型组比较,差异有统计学意义(P<0.05);模型组肾脏组织内MCP-1 mRNA的表达较正常对照组高(P<0.05),益气养阴方干预后其表达下降(P<0.05)。结论:益气养阴方能减少糖尿病肾病大鼠血糖及尿白蛋白值,该作用可能与下调MCP-1 mRNA的表达有关。