Enhancing fractalkine/CX3CR1 signalling pathway can reduce neuroinflammation by attenuating microglia activation in experimental diabetic retinopathy

The concept of diabetic retinopathy (DR) has been extended from microvascular disease to neurovascular disease in which microglia activation plays a remarkable role. Fractalkine (FKN)/CX3CR1 is reported to regulate microglia activation in central nervous system diseases. To characterize the effect of FKN on microglia activation in DR, we employed streptozotocin‐induced diabetic rats, glyoxal‐treated R28 cells and hypoxia‐treated BV2 cells to mimic diabetic conditions and explored retinal neuronal apoptosis, reactive oxygen species (ROS), as well as the expressions of FKN, Iba‐1, TSPO, NF‐κB, Nrf2 and inflammation‐related cytokines. The results showed that FKN expression declined with diabetes progression and in glyoxal‐treated R28 cells. Compared with normal control, retinal microglia activation and inflammatory factors surged in both diabetic rat retinas and hypoxia‐treated microglia, which was largely dampened by FKN. The NF‐κB and Nrf2 expressions and intracellular ROS were up‐regulated in hypoxia‐treated microglia compared with that in normoxia control, and FKN significantly inhibited NF‐κB activation, activated Nrf2 pathway and decreased intracellular ROS. In conclusion, the results demonstrated that FKN deactivated microglia via inhibiting NF‐κB pathway and activating Nrf2 pathway, thus to reduce the production of inflammation‐related cytokines and ROS, and protect the retina from diabetes insult.     

Ipriflavone as a non‐steroidal glucocorticoid receptor antagonist ameliorates diabetic cognitive impairment in mice

Diabetic cognitive impairment (DCI) is a common diabetic complication with hallmarks of loss of learning ability and disorders of memory and behavior. Glucocorticoid receptor (GR) dysfunction is a main reason for neuronal impairment in brain of diabetic patients. Here, we determined that ipriflavone (IP) a clinical anti‐osteoporosis drug functioned as a non‐steroidal GR antagonist and efficiently ameliorated learning and memory dysfunction in both type 1 and 2 diabetic mice. The underlying mechanism has been intensively investigated by assay against the diabetic mice with GR‐specific knockdown in the brain by injection of adeno‐associated virus (AAV)‐ePHP‐si‐GR. IP suppressed tau hyperphosphorylation through GR/PI3K/AKT/GSK3β pathway, alleviated neuronal inflammation through GR/NF‐κB/NLRP3/ASC/Caspase‐1 pathway, and protected against synaptic impairment through GR/CREB/BDNF pathway. To our knowledge, our work might be the first to expound the detailed mechanism underlying the amelioration of non‐steroidal GR antagonist on DCI‐like pathology in mice and report the potential of IP in treatment of DCI.     

db/db小鼠糖尿病肾病相关基因的分析和克隆

用GM-U74A基因芯片分别检测了正常对照组（db/m小鼠）、糖尿病肾病组（db/db小鼠）、大黄酸治疗组（大黄酸150 mg/kg治疗12周）肾脏基因表达谱.发现在12 437个基因（包括表达序列标签）中,与正常对照组相比,糖尿病肾病组有1 085个基因表达下调,37个基因表达上调,其中变化幅度大于2倍,表达下调的有166个和表达上调的有29个.与糖尿病肾病组相比,大黄酸治疗组有384个基因表达下调,155个表达上调,其中变化幅度大于2倍,表达下调的有47个和表达上调的有30个.在此基础上,对其中的一个差异表达的表达序列标签(EST)进行了详细的生物信息学分析,发现它是一个未知功能基因——“REKEN cDNA 0610006H10”基因的一部分.在用RT-PCR进一步验证了其与糖尿病肾病的相关性后,对“REKEN cDNA 0610006H10”基因进行了克隆.     

Differential influence of increased polyol pathway on protein kinase C expressions between endoneurial and epineurial tissues in diabetic mice

To explore the relationship between polyol pathway and protein kinase C (PKC), we examined PKC activities and expressions of PKC isoforms separately in endoneurial and vessel-rich epineurial tissues in diabetic mice transgenic for human aldose reductase (Tg). Tg and littermate control mice (Lm) were made diabetic by streptozotocin at 8 weeks of age and treated orally with aldose reductase inhibitor (ARI) (fidarestat 3-5 mg/kg/day) or placebo for 12 weeks. At the end, compared with non-diabetic state, sorbitol contents were increased 6.4-fold in endoneurium and 5.1-fold in epineurium in diabetic Tg, whereas the increase was detected only in endoneurium in diabetic Lm. Endoneurial PKC activity was significantly reduced in diabetic Tg. By contrast, epineurial PKC activity was increased in both diabetic Lm and diabetic Tg and there was no significant difference between the two groups. These changes were all corrected by ARI treatment. Consistent with the changes of PKC activities, diabetic Tg showed decreased expression of PKC alpha in endoneurium, whereas there was an increased expression of PKC beta II in epineurium in both diabetic Tg and diabetic Lm. These findings suggest the presence of dichotomous metabolic pathway between neural and vascular tissues in the polyol-PKC-related pathogenesis of diabetic neuropathy.     

Elevated glucose inhibits VEGF-A-mediated endocardial cushion formation: modulation by PECAM-1 and MMP-2

Atrioventricular (AV) septal defects resulting from aberrant endocardial cushion (EC) formation are observed at increased rates in infants of diabetic mothers. EC formation occurs via an epithelial-mesenchymal transformation (EMT), involving transformation of endocardial cells into mesenchymal cells, migration, and invasion into extracellular matrix. Here, we report that elevated glucose inhibits EMT by reducing myocardial vascular endothelial growth factor A (VEGF-A). This effect is reversed with exogenous recombinant mouse VEGF-A165, whereas addition of soluble VEGF receptor-1 blocks EMT. We show that disruption of EMT is associated with persistence of platelet endothelial cell adhesion molecule-1 (PECAM-1) and decreased matrix metalloproteinase-2 (MMP-2) expression. These findings correlate with retention of a nontransformed endocardial sheet and lack of invasion. The MMP inhibitor GM6001 blocks invasion, whereas explants from PECAM-1 deficient mice exhibit MMP-2 induction and normal EMT in high glucose. PECAM-1-negative endothelial cells are highly motile and express more MMP-2 than do PECAM-1-positive endothelial cells. During EMT, loss of PECAM-1 similarly promotes single cell motility and MMP-2 expression. Our findings suggest that high glucose-induced inhibition of AV cushion morphogenesis results from decreased myocardial VEGF-A expression and is, in part, mediated by persistent endocardial cell PECAM-1 expression and failure to up-regulate MMP-2 expression.     

Influence of vitamin E on the levels of fatty acids and MDA in some tissues of diabetic rats

This study was performed to determine whether vitamin E supplementation in streptozotocin-induced diabetic rats treated with insulin could affect the levels of fatty acid composition and malondialdehyde (MDA) of brain, liver and muscle tissues. Thirty Wistar albino rats were used during the experiments. They were randomly divided into three groups, each consisting of six individuals. The first group was diabetic, the second was control, and the third was diabetic but fed vitamin E. The level of stearic acid in brain tissues decreased (p<0.05) in the second and the third groups as compared to the first group. The percentage of arachidonic and polyunsaturated fatty acids slightly decreased (p<0.05) in the diabetic group in comparison to the second and third groups. The proportion of docosahexaenoic acid significantly increased (p<0.01) in the second and third groups in contrast to the first group. The level of docosatrienoic was slightly higher (p<0.05) in the third group than in other groups. In the liver tissues, the proportion of stearic, oleic and total monounsaturated fatty acids was slightly higher (p<0.05) in the first group than in the other groups. The level of arachidonic, docosahexaenoic, unsaturated and total polyunsaturated fatty acid slightly increased (p<0.05) in the second and third groups as compared to the first group. The level of myristic and stearic acids in muscle tissue slightly increased (p<0.05) in the first group as compared to the second and third groups. The proportion of arachidonic, docosahexaenoic and unsaturated fatty acids slightly increased (p<0.05) in the second and third groups relative to the first group. The amount of MDA was slightly higher in the diabetic group than in the other groups in all tissues. The results indicate that vitamin E supplementation, in experimental diabetes could play a role in controlling the oxidative status and altered fatty acid metabolism in tissues, thereby maintaining favourable fatty acid distribution in the tissues affected by diabetic complications.     

Regulation of mitochondrial contact sites in neonatal,juvenile and diabetic hearts

Mitochondrial contact sites (MiCS) are dynamic structures involved in high capacity transport of energy from mitochondria into the cytosole. Previous studies revealed that in normal conditions the actual number of MiCS is in correlation with the energy requirements of the heart, particularly with those for its contractile work. Although the detailed mechanisms of signalling between the processes of energy utilisation and MiCS formation in the heart are not yet elucidated, it is known that intracellular Ca²⁺ transients are intimately involved in this crosstalk. The present study is devoted to investigation of Ca²⁺-linked MiCS formation in healthy adult hearts and in hearts with modified Ca²⁺-handling such as in developing, in juvenile and diabetic myocardium. Experiments were performed on hearts of healthy rats on the 22nd embryonal day, 1st, 4th, 7th and 14th postnatal days as well as on adult hearts. Diabetic hearts were investigated on the 8th day after streptozotocin injection (45 mg.kg^–1 i.v.) to adult rats. Intracellular Ca²⁺ movements were affected by modulation of Ca²⁺ concentration in perfusion solution (1.6 or 2.2 mmol.l^–1) in isolated, Langendorff-perfused hearts, by calcium paradox (CaP) or by replacing of Ca²⁺ by Cd²⁺ ions. Elevation of extracellular Ca²⁺ was reflected by 30.1, 10.4 and 24.1% increase in intracellular free Ca²⁺ concentration in healthy adult, diabetic and 14-day old hearts respectively. In developing hearts the amount of MiCS was culminating on the 4th postnatal day. In adult hearts, elevated calcium in the perfusion solution, CaP as well as diabetes led to a significant increase in the amounts of MiCS formed (58.1, 77.2 and 86.5% respectively; p < 0.05). Diabetic and 14-day old hearts naturally exhibited amounts of MiCS comparable to those obtained by Ca²⁺-stimulation of MiCS formation in adult healthy hearts. In contrast to healthy controls, perfusion of diabetic and 14-day old hearts with elevated Ca²⁺ as well as induction of CaP exerted little influence on MiCS formation (4.4 and 8.2% for elevated Ca²⁺; 2.9 and 10.7% for CaP; p > 0.05). A replacement of Ca²⁺ by Cd²⁺ ions lowered the amount of MiCS in healthy adult and diabetic hearts (61 and 52.2%; p < 0.05). In conclusion, during development, the formation of MiCS may be influenced by both, permanent stimulation by Ca²⁺-signalling and the availability of mCPK. In healthy adult hearts the amount of MiCS is modulated by intracellular Ca²⁺ transients in response to changes in extracellular Ca²⁺ concentration. In diabetic hearts the modulation of MiCS formation is naturally attenuated, apparently as a consequence of persisting alterations in Ca²⁺-handling.     

Advanced glycation end products-induced apoptosis and overexpression of vascular endothelial growth factor in bovine retinal pericytes.

The influence of advanced glycation end products (AGEs) on apoptotic cell death and vascular endothelial growth factor (VEGF) gene expression in cultured bovine retinal pericytes was investigated. When pericytes were incubated with three immunochemically distinct AGEs, which were prepared in vitro by incubating bovine serum albumin with glucose, glyceraldehyde, or glycolaldehyde, apoptotic cell death and DNA ladder formation were significantly induced. The cytopathic effects of glyceraldehyde- or glycolaldehyde-derived AGEs were significantly enhanced in AGE receptor-transfected pericytes. Furthermore, all of these AGEs were found to upregulate the secretory forms of VEGF mRNA levels in retinal pericytes. These results suggest that AGEs disturbed retinal microvascular homeostasis by inducing pericyte apoptosis and VEGF overproduction and thus were involved in the pathogenesis of early phase diabetic retinopathy.     

糖尿病植物神经病变病人心率变异的非线性定量分析

目的：研究糖尿病人植物神经病变与心率变异的关系。对象：正常对照组和根据临床有无糖尿病神经病变（ＤＡＮ）分组的糖尿病病人,方法：应用２４小时动态心电图对正常和糖尿病人进行心率变异的线性,非线性散点图和非线性定量参数分析,结果：单纯糖尿病组ＳＤＮＮ,ＳＤＡＮＮ和ＰＮＮ５０低于正常组（Ｐ〈０．０５）;糖尿病＋ＤＡＮ组各项线性时域分析指标均低于正常和单纯糖尿病组（Ｐ〈０．０１－０．００１）,散点图分析结果