TXNIP,a novel key factor to cause Schwann cell dysfunction in diabetic peripheral neuropathy,under the regulation of PI3K/Akt pathway inhibition-induced DNMT1 and DNMT3a overexpression

Diabetic peripheral neuropathy (DPN) is the most common complication of diabetes mellitus (DM) and the dysfunction of Schwann cells plays an important role in the pathogenesis of DPN. Thioredoxin-interacting protein (TXNIP) is known as an inhibitor of thioredoxin and associated with oxidative stress and inflammation. However, whether TXNIP is involved in dysfunction of Schwann cells of DPN and the exact mechanism is still not known. In this study, we first reported that TXNIP expression was significantly increased in the sciatic nerves of diabetic mice, accompanied by abnormal electrophysiological indexes and myelin sheath structure. Similarly, in vitro cultured Schwann cells TXNIP was evidently enhanced by high glucose stimulation. Again, the function experiment found that knockdown of TXNIP in high glucose-treated RSC96 cells led to a 4.12 times increase of LC3-II/LC3-I ratio and a 25.94% decrease of cleaved caspase 3/total caspase 3 ratio. Then, DNA methyltransferase (DNMT) inhibitor 5-Aza has been reported to benefit Schwann cell in DPN, and here 5-Aza treatment reduced TXNIP protein expression, improved autophagy and inhibited apoptosis in high glucose-treated RSC96 cells and the sciatic nerves of diabetic mice. Furthermore, DNMT1 and DNMT3a upregulation were found to be involved in TXNIP overexpression in high glucose-stimulated RSC96 cells. Silencing of DNMT1 and DNMT3a effectively reversed high glucose-enhanced TXNIP. Moreover, high glucose-inhibited PI3K/Akt pathway led to DNMT1, DNMT3a, and TXNIP upregulation in RSC96 cells. Knockdown of DNMT1 and DNMT3a prevented PI3K/Akt pathway inhibition-caused TXNIP upregulation in RSC96 cells. Finally, in vivo knockout of TXNIP improved nerve conduction function, increased autophagosome and LC3 expression, and decreased cleaved Caspase 3 and Bax expression in diabetic mice. Taken together, PI3K/Akt pathway inhibition mediated high glucose-induced DNMT1 and DNMT3a overexpression, leading to cell autophagy inhibition and apoptosis via TXNIP protein upregulation in Schwann cells of DPN.Subject terms: Insulin signalling, Diabetes complications, Peripheral neuropathies     

NGF通过抑制内质网应激途径保护SCs在高糖环境诱导的凋亡

已知神经生长因子 (nerve growth factor, NGF) 对糖尿病外周神经病变 (diabetic peripheral neuropathy, DPN) 患者具有良好的治疗效果,内质网应激 (endoplasmic reticulum stress, ERS) 在调控DPN 的发生发展方面扮演着重要的角色。然而,二者间的关系未知。本研究以30 mmol/L的高糖处理RSC-96大鼠雪旺细胞 (RSC96 Schwann cells, SCs),模拟DPN患者外周神经的内环境。研究结果证实,在高糖条件下,NGF通过抑制SCs内 ERS的过度激活进而保护SCs的存活且这种抑制作用依靠P13K/AKT/GSK-3β和ERK1/2两条信号通路的调节实现。MTT检测细胞的存活率,结果显示高糖环境培养的SCs在24 h发生最佳程度的抑制,且此时间点加入的NGF浓度为50 ng/mL 时,其存活率最高。Western 印迹检测ERS和相关蛋白质的表达揭示,高糖能够过度激活SCs内ERS蛋白 (GRP-78、ATF-6、ATF-4、XBP-1、CHOP),给予 50 ng/mL的NGF处理后,ERS蛋白的表达水平大幅下调并接近正常,且此时p-AKT、p-ERK1/2、p-GSK3β蛋白的检测水平明显升高。流式细胞术检测细胞凋亡显示,NGF能显著抑制SCs的早期凋亡。上述结果证明,高糖诱导SCs的凋亡增加是由于自身的ERS被过度激活,NGF可通过调节P13K/AKT/GSK-3β和ERK1/2两条信号通路来抑制ERS的过度激活,达到保护SCs存活的目的。此机制为临床治疗 DPN 提供新的理论基础。     

Reduced cell replication and induction of apoptosis by advanced glycation end products in rat Schwann cells

We investigated the effects of advanced glycation end products (AGEs) derived from glucose, glyceraldehyde, and glycolaldehyde (designated as AGE-1, -2, and -3, respectively) on the viability, replication rate, and cytokine production of cultured Schwann cells. AGE-2 and -3, but not AGE-1, induced apoptosis, and significantly decreased the viability measured by MTT assay. The decrease was prevented completely by antioxidant alpha-lipoic acid and was prevented partially by p38 mitogen-activated protein kinase inhibitor SB202190. The decrease in mitochondrial membrane potential by AGE-2 and -3 was also observed. In addition, AGE-2 and -3 significantly suppressed the replication rate as shown by reduced bromodeoxyuridine uptake, whereas they enhanced the release of TNF-alpha and IL-1beta into the medium and activated nuclear factor-kappaB. The effects of AGE-1 on these measures were equivocal. The series of events elicited by AGE-2 and -3 may be responsible for some of the aspects of pathogenetic mechanisms in patients with diabetic neuropathy.     

Correction of protein kinase C activity and macrophage migration in peripheral nerve by pioglitazone, peroxisome proliferator activated-gamma-ligand, in insulin-deficient diabetic rats

Pioglitazone, one of thiazolidinediones, a peroxisome proliferator-activated receptor (PPAR)-γ ligand, is known to have beneficial effects on macrovascular complications in diabetes, but the effect on diabetic neuropathy is not well addressed. We demonstrated the expression of PPAR-γ in Schwann cells and vascular walls in peripheral nerve and then evaluated the effect of pioglitazone treatment for 12 weeks (10 mg/kg/day, orally) on neuropathy in streptozotocin-diabetic rats. At end, pioglitazone treatment improved nerve conduction delay in diabetic rats without affecting the expression of PPAR-γ. Diabetic rats showed suppressed protein kinase C (PKC) activity of endoneurial membrane fraction with decreased expression of PKC-α. These alterations were normalized in the treated group. Enhanced expression of phosphorylated extracellular signal-regulated kinase detected in diabetic rats was inhibited by the treatment. Increased numbers of macrophages positive for ED-1 and 8-hydroxydeoxyguanosine-positive Schwann cells in diabetic rats were also corrected by the treatment. Pioglitazone lowered blood lipid levels of diabetic rats, but blood glucose and nerve sorbitol levels were not affected by the treatment. In conclusion, our study showed that pioglitazone was beneficial for experimental diabetic neuropathy via correction of impaired PKC pathway and proinflammatory process, independent of polyol pathway.     

高糖通过Nox4型NADPH氧化酶影响施旺细胞凋亡的机制研究

目的:探讨高糖通过Nox4型NADPH氧化酶影响施旺细胞凋亡的机制。方法:提取Wistar大鼠新生鼠的施旺细胞体外培养。分为对照组、高糖组、NOX4 siRNA组及对照siRNA组(n=10)。采用WST-1法检测细胞活力,DCFH-DA法检测细胞内活性氧自由基(ROS)含量,荧光实时定量RT-PCR检测Nox4和Caspase3 mRNA表达,蛋白印迹法检测Nox4和Caspase3蛋白表达。结果:高糖培养上调施旺细胞Nox4 mRNA及蛋白表达,降低施旺细胞活性,增加细胞内ROS含量,通过增加Caspase3 mRNA及蛋白表达促进细胞凋亡。NOX4 siRNA通过抑制Nox4基因表达,阻止高糖培养的施旺细胞内ROS蓄积,降低高糖对施旺细胞的活性损害,通过下调Caspase3 mRNA及蛋白表达减少细胞凋亡。结论:Nox4参与高糖引起的施旺细胞凋亡,针对Nox4表达或功能的调控方式可能成为治疗糖尿病周围神经病变的新途径。     

The organotellurium compound ammonium trichloro(dioxoethylene-0,0') tellurate enhances neuronal survival and improves functional outcome in an ischemic stroke model in mice

Ammonium trichloro(dioxoethylene-0,0') tellurate (AS101) is a non-toxic organotellurium compound with pleiotropic activities. It was recently shown to induce production of the neurotrophic factor glial cell line-derived neurotrophic factor and to rescue neuronal-like PC-12 cells from neurotrophic factor deprivation-induced apoptosis. In this study, we show that AS101 improves functional outcome and reduces brain damage in a mouse model of focal ischemic stroke. Both pre-stroke and post-stroke intraperitoneal treatments with AS101 reduced infarct size and edema and improved the neurological function of the animals. AS101 treatments reduced both apoptotic and inflammatory caspase activities, and also inhibited protein tyrosine nitration suggesting that AS101 suppresses oxidative stress. Studies of cultured neurons showed that AS101 confers protection against apoptosis induced by either glucose deprivation or the lipid peroxidation product 4-hydroxynonenal. Moreover, AS101 treatment reduced glutamate-induced intracellular calcium elevation, a major contributor to neuronal death in stroke. As AS101 has an excellent safety profile in humans, our pre-clinical data suggest a potential therapeutic benefit of AS101 in patients suffering from stroke and other neurodegenerative conditions.     

Effects of Glucose, Insulin, and Supernatant from Pancreatic β-cells on Brain–Pancreas Relative Protein in Rat Hippocampus

Brain–pancreas relative protein (BPRP) is a novel protein that mainly expresses in brain and pancreas. In our previous study, we found that various stressors significantly decreased the expression of BPRP in pancreas in vivo, accompanied by changes in insulin and glucose levels, and that expression of BPRP in pancreas also decreased significantly in diabetic rats induced by Streptozocin (STZ). All these findings suggest that BPRP may be a glucose or insulin-sensitive protein. However, how the changes in insulin or glucose levels influence the expression of BPRP in hippocampus requires further study. Here, we investigated the effects of insulin or glucose on the expression of BPRP in primary cultured hippocampal neurons. We supplied hippocampal neurons with glucose, insulin, or supernatant from pancreatic β-cells, which secrete insulin into the supernatant. Our data showed that insulin had beneficial effect on the viability while no significant effect on the expression of BPRP in hippocampal neurons. On the contrary, 40 mM glucose or free glucose culture significantly decreased the expression of BPRP, while had no significant effect on the viability and apoptosis of hippocampal neurons. Further study showed that levels of insulin in the supernatant collected from pancreatic β-cells medium changed over days, and that supernatant increased the viability of hippocampal neurons, while it had no obvious effect on the expression of BPRP in hippocampal neurons. These results suggest that BPRP may be a glucose-sensitive protein.     

Overexpression of STAMP2 suppresses atherosclerosis and stabilizes plaques in diabetic mice

Our research aims to evaluate the function of the STAMP2 gene, an important trigger in insulin resistance (IR), and explore its role in macrophage apoptosis in diabetic atherosclerotic vulnerable plaques. The characteristics of diabetic mice were measured by serial metabolite and pathology tests. The level of STAMP2 was measured by RT‐PCR and Western blot. The plaque area, lipid and collagen content of brachiocephalic artery plaques were measured by histopathological analyses, and the macrophage apoptosis was measured by TUNEL. Correlation of STAMP2/Akt signaling pathway and macrophage apoptosis was validated by Ad‐STAMP2 transfection and STAMP2 siRNA inhibition. The diabetic mice showed typical features of IR, hyperglycaemia. Overexpression of STAMP2 ameliorated IR and decreased serum glucose level. In brachiocephalic lesions, lipid content, macrophage quantity and the vulnerability index were significantly decreased by overexpression of STAMP2. Moreover, the numbers of apoptotic cells and macrophages in lesions were both significantly decreased. In vitro, both mRNA and protein expressions of STAMP2 were increased under high glucose treatment. P‐Akt was highly expressed and caspase‐3 was decreased after overexpression of STAMP2. However, expression of p‐Akt protein was decreased and caspase‐3 was increased when STAMP2 was inhibited by siRNA. STAMP2 overexpression could exert a protective effect on diabetic atherosclerosis by reducing IR and diminishing macrophage apoptosis.     

Hyperglycemia triggers abnormal signaling and proliferative responses in Schwann cells

Peripheral neuropathy is a serious diabetic complication. Delayed nerve regeneration in diabetic animal models suggests abnormalities in proliferation/differentiation of Schwann cells (SC). We recently reported that endothelins (ETs) regulate proliferation and phenotype in primary and immortalized SC (iSC). We now investigated changes in the effects of ETs on SC proliferation and signaling in nerve segments from streptozotocin-induced diabetic rats and in iSC exposed to high glucose. Cultured explants from diabetic rats displayed a delay in the time-course of [³H]-thymidine incorporation as well as enhanced sensitivity to endothelin-1 (ET-1) or insulin. iSC cultured in high (25 mM) glucose-containing media also exhibited higher [³H]-thymidine incorporation, along with an enhanced activation of p38 mitogen-activated protein kinase and phospholipase C in response to ET-1 or platelet-derived growth factor as compared to controls (5.5 mM glucose). These studies support an extra-vascular role of ETs in peripheral nerves and SC. The increased sensitivity to ET-1 in nerves and iSC exposed to high glucose may contribute to abnormal SC proliferation characterizing diabetic neuropathy.     

Effect of Sorbinil and Ascorbic Acid on myo-Inositol Transport in Cultured Rat Schwann Cells Exposed to Elevated Extracellular Glucose

Abstract: The effect of long-term (2 weeks) exposure to 0–50 m M glucose and 0–1 m M sorbitol on myo -inositol metabolism was studied in cultured rat Schwann cells. Experiments were carried out to determine the effect of sorbinil and ascorbic acid on myo -inositol uptake in rat Schwann cells cultured in the presence of increased extracellular glucose or sorbitol. myo -Inositol uptake and its incorporation into phospholipids decreased significantly when cells were grown in ≥30 m M glucose for a period of 2 weeks. This inhibitory effect was partly blocked by sorbinil, an aldose reductase inhibitor, in a dose-dependent fashion. Significant prevention was achieved with 0.5 and 1 m M sorbinil. Ascorbic acid also prevented the reduction in myo -inositol uptake due to excess extracellular glucose, at 3 and 30 µ M concentrations, but not at 300 µ M . Neither sorbinil nor ascorbic acid could prevent the alterations in myo -inositol transport in cells exposed to high sorbitol levels for the same period of time. These data suggest that glucose-induced alteration of myo -inositol transport in Schwann cells is mediated, at least in part, via sorbitol accumulation. This myo -inositol transport impairment is prevented by sorbinil and also by ascorbic acid. Ascorbic acid may hold a fresh promise for the treatment/prevention of diabetic neuropathy/complications, at least as an adjunct therapy along with known aldose reductase inhibitors.     

Flavonoid compound breviscapine suppresses human osteosarcoma Saos‐2 progression property and induces apoptosis by regulating mitochondria‐dependent pathway

This study was aimed to investigate the ability of a flavonoid compound breviscapine (BVP) to suppress growth and elicit apoptosis in human osteosarcoma (OS) Saos‐2 cells. The cells were cultured in vitro and treated with three concentrations of BVP (80, 160, and 320 μg/ml). Moreover, C57 mice were injected with Saos‐2 cells to establish a subcutaneous xenograft model, and they were subsequently treated with three doses of BVP via intraperitoneal injection. The viability of the cells was examined by the Cell Counting Kit‐8 method. The apoptotic cells were assessed by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. The tumor volume and weight were monitored from day 3 through day 21 after the last injection. The expression of bax, bcl‐2, and cytochrome c (cyt c) mRNA was detected by a real‐time polymerase chain reaction. The protein levels of bax, bcl‐2, cyt c, caspase 3, and caspase 9 were evaluated by Western blot. The expression and distribution of bcl‐2 and bax in tissues were detected by immunohistochemistry. Compared with the control group, BVP treatment inhibited cell proliferation and induced apoptosis of Saos‐2 cells in vitro. Consistently, treatment of mice bearing transplanted tumors with BVP suppressed the growth of OS tumors and promoted cell apoptosis; it also reduced tumor volume and weight. Mechanistically, BVP‐induced apoptosis was mediated by the mitochondria‐dependent pathway, as evidenced by the increased expression of bax and cyt c and the decreased expression of bcl‐2, as well as activation of caspase 9 and caspase 3 in vitro and in vitro. Collectively, BVP inhibits growth and promotes apoptosis of OS by activating the mitochondrial apoptosis pathway.     

Cx31.1 expression is modulated in HaCaT cells exposed to UV‐induced damage and scrape‐wounding

Connexin31.1 (Cx31.1) is a gap junction protein associated with apoptosis. In the skin, apoptosis is modulated by diabetes. A HaCaT skin model investigated whether normal (NGI) and high glucose and insulin (HGI; diabetic) conditions altered Cx31.1 expression, and if these were apoptosis linked. Cx31.1 was found in HaCaT and HeLa Ohio cells, with HaCaT Cx31.1 protein increased in HGI conditions, and around apoptotic cells. HeLa Cx31.1 channels were noncommunicative. Post scrape‐wounding, Cx31.1 increased at wound edges. Caspase 3/7 in scrape‐wounds media (containing cells) elevated in HGI. UV exposure raised Cx31.1, and caspase 3/7, in NGI and HGI. UV reduced cell viability in NGI cells, although not significantly in HGI. Cx31.1 is modulated during HaCaT cell wound closure, and associated with ‘diabetic’ conditions. Cx31.1 expression matched apoptosis levels, higher in HGI cultures. Cx31.1 is noncommunicating, modulated after wounding, linked to apoptosis, and may be associated with tissue turn‐over around diabetic wounds.     

雌激素处理的hBMSC对高糖诱导血管内皮细胞损伤的保护作用*

目的: 探讨雌激素处理人骨髓间充质干细胞(hBMSC)对高糖诱导的人脐静脉血管内皮细胞(HUVEC)损伤的保护作用及机制。方法: 采用30 mmol/L葡萄糖刺激hBMSC细胞建立高糖模型并分组:以无刺激者为高糖对照组(HG组)、以20 μmol/L雌激素处理者为高糖雌激素组(HG+E2组)、以5 μmo/L蛋白激酶B(PKB/Akt)抑制剂Triciribine预处理45 min后,再以20 μmol/L雌激素处理者为高糖Akt抑制剂组(HG+E2+Triciribine组)和正常条件培养的hBMSC为正常对照组(NG组)。分别于处理12 h后,采用CCK8法检测各组hBMSC的细胞活力,硝酸还原酶法和ELISA法检测各组培养基上清中NO、VEGF和IL-8的含量(n=6),48 h后采用Western blot检测内皮型一氧化氮合酶(eNOS)和磷酸化eNOS(p-eNOS)蛋白表达水平(n=3)。此外,提取各组hBMSC的培养基上清作为条件培养基(CM)培养人脐静脉血管内皮细胞(HUVEC)并分组为:HG-CM组(HG组条件培养基处理)、HG+E2-CM组(HG+E2组条件培养液处理)、HG+E2+Triciribine-CM组(HG+E2+Triciribine组条件培养基处理)和HG-H组(高糖对照组,30 mmol/L葡萄糖终浓度处理),分别于12 h后,采用CCK8法检测各组HUVEC的细胞活力(n=6),24 h后采用流式细胞术检测各组HUVEC细胞的凋亡率(n=3);48 h后采用划痕实验观察各组HUVEC细胞的迁移率(n=3)。结果: 与NG组相比,HG组中hBMSC细胞活力和细胞内eNOS蛋白磷酸化水平降低(P<0.05),细胞培养液上清中NO、VEGF和IL-8含量减少(P<0.05);与HG组相比,HG+E2组中hBMSC的细胞活力和细胞中eNOS蛋白磷酸化水平显著增加(P<0.05),细胞培养基上清中NO、VEGF和IL-8含量增加(P<0.05),而当hBMSC细胞中Akt蛋白活性被抑制后,HG+E2+Triciribine组中上述结果指标呈反向变化(P<0.05)。此外,与HG-CM组相比,HG+E2-CM组中HUVECs的细胞活力和迁移能力显著增加(P<0.05),细胞凋亡比例降低(P<0.05),而与HG+E2-CM组相比,HG+E2+Triciribine-CM组中HUVECs的细胞活力和迁移能力降低(P<0.05),细胞凋亡比例增加(P< 0.05)。结论: 雌激素可能通过激活hBMSC细胞Akt/eNOS信号通路,促进NO、VEGF和IL-8的分泌,进而增加HUVECs的细胞活力和迁移能力,并抑制细胞凋亡的发生,对高糖诱导的HUVECs细胞损伤发挥保护作用。     

The TRPV1 receptor is associated with preferential stress in large dorsal root ganglion neurons in early diabetic sensory neuropathy

Chronic diabetic neuropathy is associated with peripheral demyelination and degeneration of nerve fibers. The mechanism(s) underlying neuronal injury in diabetic sensory neuropathy remain poorly understood. Recently, we reported increased expression and function of transient receptor potential vanilloid 1 (TRPV1) in large dorsal root ganglion (DRG) neurons in diabetic sensory neuropathy. In this study, we examined the effects of TRPV1 activation on cell injury pathways in this subpopulation of neurons in the streptozotocin-induced diabetic rat model. Large DRG neurons from diabetic (6–8 weeks) rats displayed increased oxidative stress and activation of cell injury markers compared with healthy controls. Capsaicin (CAP) treatment induced decreased labeling of MitoTracker Red and increased cytosolic cytochrome c and activation of caspase 3 in large neurons isolated from diabetic rats. CAP treatment also induced oxidative stress in large diabetic DRG neurons, which was blocked by pre-treatment with caspase or calpain inhibitor. In addition, both μ-calpain expression and calpain activity were significantly increased in DRG neurons from diabetic rats after CAP treatment. Treatment with capsazepine, a competitive TRPV1 antagonist, markedly reduced these abnormalities in vitro and prevented activation of cell injury in large DRG neurons in diabetic rats in vivo . These results suggest that activation of the TRPV1 receptor activates pathways associated with caspase-dependent and calpain-dependent stress in large DRG neurons in STZ-diabetic rats. Activation of the TRPV1 receptor may contribute to preferential neuronal stress in large DRG neurons relatively early in diabetic sensory neuropathy.     

Astragaloside IV alleviates Schwann cell injury in diabetic peripheral neuropathy by regulating microRNA-155-mediated autophagy

BackgroundMicroRNA-155(miR-155) is closely associated with diabetic peripheral neuropathy (DPN). Astragaloside IV (AST) is a significant extract of Astragalus membranaceus, which has been found to be effective in the treatment of DPN. However, whether astragaloside IV alleviate DPN via regulating miR-155-mediated autophagy remains unclear.PurposeThis study was designed to evaluate the effects of AST on DPN myelin Schwann cells injury and explore the mechanism of AST in treating DPN for the first time.MethodsGK rats fed with high-fat diet and RSC96 cells cultured in high glucose were used to establish DPN Schwann cells injury in vivo and in vitro model. The effects of AST on DPN were explored through blood glucose detection, nerve function detection, pathological detection and the expression of Neuritin detected by immunohistochemical. To study the effect of AST on the DPN Schwann cells autophagy and the upstream PI3K/Akt/mTOR pathway, the expressions of beclin-1 and LC3 were detected by western blot (WB) in sciatic nerves and by immunofluorescence (IFC) in RSC96 cells. The real-time polymerase chain reaction (RT-PCR) was applied to detect the expressions of miR-155, ATG5, ATG12 both in vivo and in vitro. The binding effect of miR-155 and target gene PI3KCA was verified by luciferase reporter gene assay. The expressions of PI3K, p-Akt/Akt, p-mTOR/mTOR were detected by WB and the expressions of PI3KCA were detected by RT-PCR in vitro. The apoptosis was detected by flow cytometry. Meanwhile, the influence of miR-155 overexpression and knocked down on the above indicators was also detected in RSC96 cells. At last, further mechanism experiments were conducted to verify the mechanism of AST regulating the autophagy and apoptosis of RSC96 cells.ResultsAST reduced blood glucose levels, alleviated peripheral nerve myelin sheath injury, and improved neurological function in DPN rats. In addition, AST enhanced the autophagy activity and alleviated the apoptosis in RSC96 cell. Mechanism study shown that AST promote autophagy via regulating miR-155-mediated PI3K/Akt/mTOR signaling pathways. AST reduced RSC96 cells apoptosis by promoting autophagy.ConclusionAST alleviate the myelin sheath injury of DPN caused by the apoptosis of Schwann cells via enhancing autophagy, which was attributed to inhibiting the activation of the PI3K/Akt/mTOR signaling pathway by upregulating miR-155 expression.     

Protective effects of intracellular reactive oxygen species generated by 6-formylpterin on tumor necrosis factor-alpha-induced apoptotic cell injury in cultured rat hepatocytes

The effects of 6-formylpterin on tumor necrosis factor (TNF)-alpha-induced apoptotic cell injury were studied in cultured rat hepatocytes. The incubation of the hepatocytes with TNF-alpha and actinomycin D (ActD) induced the apoptotic cell injury. The level of aspartate transaminase (AST) in the culture supernatant increased, and the cell viability, estimated by mitochondrial respiration (MTT assay), decreased. The DNA fragmentation and the caspase 3-like activity, which are characterized to apoptosis, increased. When the hepatocytes were incubated with 100-500 microM 6-formylpterin, the intracellular formation of reactive oxygen species (ROS) was observed, and the ratio of reduced and oxidized glutathione (GSH/GSSG) of whole cell lysate decreased. The co-incubation of the TNF-alpha/ActD-treated hepatocytes with 100-500 microM 6-formylpterin attenuated the TNF-alpha/ActD-induced apoptotic cell injury. The level of AST decreased and the cell viability increased. Both the DNA fragmentation and the caspase 3-like activity decreased. The caspases, executors of apoptosis, are known to require a reduced cystein in their active site to function, and the intact intracellular GSH/GSSG is essential for the caspase activation. Therefore, our findings suggest that intracellular ROS generated by 6-formylpterin decline the intracellular redox state to an oxidant state, which suppresses the caspase activity and prevents the apoptotic cell injury of hepatocytes.     

Caspase‐8: a key role in the pathogenesis of diabetic embryopathy

Maternal diabetes causes neural tube defects in embryos, which are associated with increased apoptosis in the neuroepithelium. Many factors, including effector caspases, have been shown to be involved in the events. However, the key regulators have not been identified and the underlying mechanisms remain to be addressed. Caspase‐8, an initiator caspase, has been shown to be altered in diabetic embryopathy, suggesting a role as an upstream apoptotic regulator. Using mouse embryos as a model system, this study demonstrates that caspase‐8 is required for the production of hyperglycemia‐associated embryonic malformations. Caspase‐8 was shown to be expressed in the developing neural tube. Its activity, as evidenced by enhanced cleavage, was increased by hyperglycemia. These changes were associated with increased formation of the active cleavage of Bid. Inhibition of caspase‐8 activity in high glucose–challenged embryos reduced the rate of embryonic malformation and this was associated with decreased apoptosis in the neuroepithelium of the neural tube. Inhibition of caspase‐8 activity also reduced hyperglycemia‐induced Bid activation and caspase‐9 cleavage. These data suggest that caspase‐8 may control diabetic embryopathy‐associated apoptosis via regulation of the Bid‐stimulated mitochondrion/caspase‐9 pathway. Birth Defects Res (Part B)86:72‐77, 2009. ©2009 Wiley‐Liss, Inc.     

Neuronal death in primary retinal cultures is related to nitric oxide production, and is inhibited by erythropoietin in a glucose-sensitive manner

The aim of this work was to investigate the interrelated effects of glucose, nitric oxide (NO) and erythropoietin on neuronal survival in retinal cultures, thereby exploring the mechanism of neuronal death in the diabetic retina. Rat retinal cells were cultured in low (5 mM) or high (15 mM) glucose concentrations. After 9 days, cell viability was assessed by (3,4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and NO production was determined by the Griess reaction. Immunohistochemistry was used to quantify GABA-labelled neurones and cells staining for DNA breakdown. High or low glucose concentrations had no effect on basal NO production or the survival of neurones in culture, but treatment with N-nitro-L-arginine methyl ester reduced extracellular levels of NO and increased neuronal survival at both concentrations of glucose. Erythropoietin decreased cell death and NO levels, but only in cultures grown in low concentrations of glucose. It is concluded that erythropoietin's neurotrophic function in the retina is attenuated at glucose concentrations similar to those which occur in diabetes.     

Effects of mechanical vibration on cell morphology,proliferation, apoptosis,and cytokine expression/secretion in osteocyte‐like MLO‐Y4 cells exposed to high glucose

Diabetic patients exhibit significant bone deterioration. Our recent findings demonstrate that mechanical vibration is capable of resisting diabetic bone loss, whereas the relevant mechanism remains unclear. We herein examined the effects of mechanical vibration on the activities and functions of osteocytes (the most abundant and well‐recognized mechanosensitive cells in the bone) exposed to high glucose (HG). The osteocytic MLO‐Y4 cells were incubated with 50 mM HG for 24 h, and then stimulated with 1 h/day mechanical vibration (0.5 g, 45 Hz) for 3 days. We found that mechanical vibration significantly increased the proliferation and viability of MLO‐Y4 cells under the HG environment via the MTT, BrdU, and Cell Viability Analyzer assays. The apoptosis detection showed that HG‐induced apoptosis in MLO‐Y4 cells was inhibited by mechanical vibration. Moreover, increased cellular area, microfilament density, and anisotropy in HG‐incubated MLO‐Y4 cells were observed after mechanical vibration via the F‐actin fluorescence staining. The real‐time polymerase chain reaction and western blotting results demonstrated that mechanical vibration significantly upregulated the gene and protein expression of Wnt3a, β‐catenin, and osteoprotegerin (OPG) and decreased the sclerostin, DKK1, and receptor activator for nuclear factor‐κB ligand (RANKL) expression in osteocytes exposed to HG. The enzyme‐linked immunosorbent assay assays showed that mechanical vibration promoted the secretion of prostaglandin E₂ and OPG, and inhibited the secretion of tumor necrosis factor‐α and RANKL in the supernatant of HG‐treated MLO‐Y4 cells. Together, this study demonstrates that mechanical vibration improves osteocytic architecture and viability, and regulates cytokine expression and secretion in the HG environment, and implies the potential great contribution of the modulation of osteocytic activities in resisting diabetic osteopenia/osteoporosis by mechanical vibration.     

Neuroprotective effects of astaxanthin against oxygen and glucose deprivation damage via the PI3K/Akt/GSK3β/Nrf2 signalling pathway in vitro

Astaxanthin (ATX), which is the most abundant flavonoid in propolis, has previously shown neuroprotective properties against cerebral ischaemia‐induced apoptosis. However, the mechanisms by which ATX mediates its therapeutic effects are unclear. At present, we explored the underlying mechanisms involved in the protective effects of ATX via the phosphoinositide 3‐kinase (PI3K)/Akt/glycogen synthase kinase 3 beta (GSK3β)/nuclear factor erythroid 2‐related factor 2 (Nrf2) signalling pathway in SH‐SY5Y cells. The PI3K/Akt inhibitor LY294002 and GSK3β inhibitor LiCl were employed in this study. Pre‐treatment with ATX for 24 hours significantly decreased the oxygen and glucose deprivation (OGD)‐induced viability loss, reduced the proportion of apoptosis and regulated OGD‐mediated reactive oxygen species (ROS) production. Furthermore, ATX suppressed OGD‐caused mitochondrial membrane potential and decomposition of caspase‐3 to cleaved caspase‐3, and heightened the B‐cell lymphoma 2 (Bcl‐2)/Bax ratio. PI3K/Akt/GSK3β/Nrf2 signalling pathway activation in SH‐SY5Y cells was verified by Western blot. ATX and LiCl treatment raised the protein levels of p‐Akt, p‐GSK3β, nucleus Nrf2 and haeme oxygenase 1 (HO‐1). However, these protein expression levels decreased by treatment of LY294002. The above in vitro data indicate that ATX can confer neuroprotection against OGD‐induced apoptosis via the PI3K/Akt/GSK3β/Nrf2 signalling pathway.