PDGF-β receptor and PKC have no effect on angiotensin II-induced JAK2 and STAT1 phosphorylation in vascular smooth muscle cells under high glucose condition

Background: The mechanisms responsible for the accelerated cardiovascular disease in diabetes, as well as the increased hypertrophic effects of angiotensin II (Ang II) under hyperglycemic condition, are not very clear. Evidences show that platelet-derived growth factor (PDGF) and protein kinase C (PKC) play a critical role in this effect. In our study, we examined the role of PKC and PDGF receptor on JAK2 and STAT1 phosphorylation under high glucose (HG) condition (25 mmol/L) in response to Ang II in cultured vascular smooth muscle cells (VSMC).

Methods: VSMCs were isolated from the thoracic aorta of male Wistar rats and were cultured. Growth-arrested VSMCs were placed in either normal glucose (NG) or HG condition for 48?h and then VSMCs were stimulated with agonists and antagonists. The tyrosine phosphorylation of JAK2 or STAT were determined by immunoblotting using specific antibodies.

Results: High glucose markedly increased the phosphorylation of tyrosine residues of JAK2 and serine residues of STAT 1 compared with cells cultured in NG (5.5 mmol/L) with and without Ang II stimulation. Experiments made with specific PDGF-β receptor inhibitor AG1295 and PKC inhibitor GF109203X showed that there were no changes in Ang II-stimulated JAK2 and STAT1 phosphorylation under NG and HG conditions compared with experiments without inhibitors.

Conclusion: According to our findings, Ang II-stimulated JAK2 and STAT1 phosphorylation under either NG or HG condition do not proceed via a different pathway rather than PKC and PDGF-β receptor.     

Time‐lapse microscopy of oxidative stress demonstrates metabolic sensitivity of retinal pericytes under high glucose condition

Hyperglycemia affects retinal vascular cell function, promotes the development and progression of diabetic retinopathy and ultimately causes vision loss. Oxidative stress, reactive oxygen species (ROS) in excess, is a key biomarker for diabetic retinopathy. Using time‐lapse fluorescence microscopy, ROS dynamics was monitored and the metabolic resistivity of retinal endothelial cells (REC) and pericytes (RPC) was compared under metabolic stress conditions including high glucose (HG). In the presence of a mitochondrial stressor, REC exhibited a significant increase in the rate of ROS production compared with RPC. Thus, under normal glucose (NG), REC may utilize oxidative metabolism as the bioenergetic source, while RPC metabolic activity is independent of mitochondrial respiration. In HG condition, the rate of ROS production in RPC was significantly higher, whereas this rate remained unchanged in REC. Thus, under HG condition RPC may preferentially utilize oxidative metabolism, which results in increased rate of ROS production. In contrast, REC use glycolysis as their major bioenergetic source for ATP production, and consequently HG minimally affects their ROS levels. These observations are consistent with our previous studies where we showed HG condition has minimal effect on apoptosis of REC, but results in increased rate of apoptosis in RPC. Collectively, our results suggest that REC and RPC exhibit different metabolic activity preferences under different glucose conditions. Thus, protection of RPC from oxidative stress may provide an early point of intervention in development and progression of diabetic retinopathy.      

Metformin induces suppression of NAD(P)H oxidase activity in podocytes

Hyperglycemia increases the production of reactive oxygen species (ROS). NAD(P)H oxidase, producing superoxide anion, is the main source of ROS in diabetic podocytes and their production contributes to the development of diabetic nephropathy. We have investigated the effect of an antidiabetic drug, metformin on the production of superoxide anion in cultured podocytes and attempted to elucidate underlying mechanisms.The experiments were performed in normal (NG, 5.6 mM) and high (HG, 30 mM) glucose concentration. Overall ROS production was measured by fluorescence of a DCF probe. Activity of NAD(P)H oxidase was measured by chemiluminescence method. The AMP-dependent kinase (AMPK) activity was determined by immunobloting, measuring the ratio of phosphorylated AMPK to total AMPK. Glucose accumulation was measured using 2-deoxy-[1,2-³H]-glucose.ROS production increased by about 27% (187 ± 8 vs. 238 ± 9 arbitrary units AU, P < 0.01) in HG. Metformin (2 mM, 2 h) markedly reduced ROS production by 45% in NG and 60% in HG. Metformin decreased NAD(P)H oxidase activity in NG (36%) and HG (86%). AMPK activity was increased by metformin in NG and HG (from 0.58 ± 0.07 to. 0.99 ± 0.06, and from 0.53 ± 0.03 to 0.64 ± 0.03; P < 0.05). The effects of metformin on the activities of NAD(P)H oxidase and AMPK were abolished in the presence of AMPK inhibitor, compound C.We have shown that metformin decreases production of ROS through reduction of NAD(P)H oxidase activity. We also have demonstrated relationship between activity of NAD(P)H oxidase and AMPK.     

High glucose augments the angiotensin II-induced activation of JAK2 in vascular smooth muscle cells via the polyol pathway

Angiotensin II (Ang II), protein kinase C (PKC), reactive oxygen species (ROS) generated by NADPH oxidase, the activation of Janus kinase 2 (JAK2), and the polyol pathway play important parts in the hyperproliferation of vascular smooth muscle cells (VSMC), a characteristic feature of diabetic macroangiopathy. The precise mechanism, however, remains unclear. This study investigated the relation between the polyol pathway, PKC-beta, ROS, JAK2, and Ang II in the development of diabetic macroangiopathy. VSMC cultured in high glucose (HG; 25 mm) showed significant increases in the tyrosine phosphorylation of JAK2, production of ROS, and proliferation activities when compared with VSMC cultured in normal glucose (5.5 mm (NG)). Both the aldose reductase specific inhibitor (zopolrestat) or transfection with aldose reductase antisense oligonucleotide blocked the phosphorylation of JAK2, the production of ROS, and proliferation of VSMC induced by HG, but it had no effect on the Ang II-induced activation of these parameters in both NG and HG. However, transfection with PKC-beta antisense oligonucleotide, preincubation with a PKC-beta-specific inhibitor (LY379196) or apocynin (NADPH oxidase-specific inhibitor), or electroporation of NADPH oxidase antibodies blocked the Ang II-induced JAK2 phosphorylation, production of ROS, and proliferation of VSMC in both NG and HG. These observations suggest that the polyol pathway hyperactivity induced by HG contributes to the development of diabetic macroangiopathy through a PKC-beta-ROS activation of JAK2.     

High Glucose-enhanced Acetylcholine Stimulated CGMP Masks Impaired Vascular Reactivity in Tail Arteries from Short-Term Hyperglycemic Rats

Impaired vascular endothelium-dependent relaxation and augmented contractile responses have been reported in several models of long-term hyperglycemia. However, the effects of short-term ambient hyperglycemia are poorly understood. Since oxidative stress has been implicated as a contributor to impaired vascular function, we investigated the following:Aims: (1) the effects of high glucose exposure in vitro (7 – 10 days) on vascular relaxation to acetylcholine (Ach) and contractility to norepinephrine (NE) and KCl; (2) if NO-dependent cGMP generation is affected under these conditions; and (3) aortic redox status.Methods: Non-diabetic rat tail artery rings were incubated in normal (5mM) (control NG) or high (20mM) glucose buffer (control HG). Vascular responses to Ach, NE and KCl were compared to those of streptozotocin (SZ) diabetic animals in the same buffers (diabetic NG, diabetic HG). Ach stimulated cGMP levels were quantitated as an indirect assessment of endothelial nitric oxide (NO) production and oxidative stress evaluated by measuring vascular glutathione and oxidized glutathione.Results: Rings from diabetic rats in NG showed impaired relaxation to Ach (P = 0.002) but relaxed normally, when maintained in HG. Similarly, contractile responses to NE were attenuated in diabetic rings in NG but similar to controls in HG. HG markedly augmented maximal contraction to KCl compared to control and diabetic vessels in NG (P < 0.0001). Diabetic vessels in a hyperosmolar, but normoglycemic, milieu respond like those in HG. in vitro, HG for 2 hours changed neither relaxation nor contractile responses to NE and KCl in control rings. Basal cGMP levels were lower in aortae from diabetic animals pre-incubated in NG than in HG/LG or in control rings in NG (P < 0.05). cGMP responses to Ach were exaggerated in diabetic vessels in HG (P = 0.035 vs. control NG, P = 0.043 vs. diabetic NG) but not different between control and diabetic rings in NG. Vessels from diabetic animals had lower levels of GISH (P < 0.0001) and higher levels of GSSG (P < 0.0001) indicating oxidative stress.Conclusions: Our data indicate that endothelium dependent relaxation is altered early in the diabetic state and that increased NO responses may compensate for augmented oxidative stress but the lack of effect of short-term exposure of normal vessels to HG suggests that short-term hyperglycemia per se does not cause abnormal vascular responses.     

Adipose-derived mesenchymal stromal cells reverse high glucose–induced reduction of angiogenesis in human retinal microvascular endothelial cells

Background aimsDiabetic retinopathy (DR) is characterized by a progressive alteration of the retinal microvasculature, arising from microaneurysms to leaky vessels and finally abnormal neovascularization. The hyperglycemia-mediated loss of pericytes is a key event in vessel degeneration causing vascular destabilization. To overcome this, mesenchymal stromal cells (MSCs) have been tested as pericyte replacement in several animal models showing repair and regeneration of DR-damaged vasculature.MethodsWe hypothesized that adipose-derived mesenchymal stromal cells (ASCs) resist high glucose–induced challenges and protect human retinal microvascular endothelial cells (HRMVECs) from glucose-mediated injury. ASCs and HRMVECs were cultured under normal-glucose (NG; 1 g/L) and high-glucose (HG; 4.5 g/L) conditions comparing their phenotype and angiogenic potential.ResultsWhereas ASCs were generally unaffected by HG, HG caused a reduction of the angiogenic potential in HRMVEC. Indeed, HG-treated HRMVECs formed fewer vascular tube structures in a basement membrane angiogenesis assay. However, this was not observed in a direct ASC and HRMVEC coculture angiogenesis assay. Increased oxidative stress levels appeared to be linked to the HG-induced reduction of angiogenesis, which could be restored by ASC-conditioned medium and antioxidant treatment.ConclusionsThese findings suggest that ASC resist HG-stress whereas endothelial cell angiogenic capacity is reduced. Thus, ASC may be potentially therapeutically active in DR by restoring angiogenic deficits in retinal endothelial cells by the secretion of proangiogenic factors. However, these data also inquire for a thorough risk assessment about the timing of the ASC-based cell therapy, which can be considered advantageous at early stage of DR, but possibly detrimental at the late neo-angiogenic stage of DR.     

Protective effects of Da-cheng-qi decoction in rats with intracerebral hemorrhage

BackgroundIntracerebral hemorrhage (ICH), the most fatal subtype of stroke, has no disease-modifying treatment. Da-cheng-qi decoction (DCQ), composed of rhubarb, is one of the most commonly used Chinese traditional decoctions in ICH treatment. But the mechanism is not clear. Emodin is an active compound found in rhubarb.PurposeTo study the protective effects of DCQ on ICH and its possible mechanisms of action.MethodsThe ICH model was reproduced by injecting collagenase-VII into the left caudate putamen (CPu) of rats. DCQ and emodin were used to treat the ICH rats for 7 days. Behavior tests, proteomic analysis, morphological studies, and western blotting were performed.ResultsThe neurological deficits in the ICH rats recovered with DCQ and emodin on the 14^th day after ICH. The proteomics data revealed that DCQ significantly corrected the pathological signals in the CPu and hippocampus after ICH. The numbers of amoebic microglia in the CPu and M2 microglia in both CPu and hippocampus were significantly increased after DCQ and emodin treatment. The increase in GluN2B-containing NMDA receptor (NR2B) and postsynaptic density protein-95, activation of mitogen-activated protein kinase (MAPK) signals in the CPu, and secondary neurodegeneration (SND) in the hippocampus were significantly recovered in DCQ-treated rats. Inhibition of MAPK p38 (p38) in the hippocampus was observed after DCQ and emodin treatment.ConclusionThe protective effects of DCQ on ICH were confirmed in this study, and its mechanism may be related to the inhibition of MAPK and activation of M2 microglia. These results are beneficial to the development of ICH therapeutic targets.     

Glucosamine regulates hepatic lipid accumulation by sensing glucose levels or feeding states of normal and excess

Dose-dependent lipid accumulation was induced by glucose in HepG2 cells. GlcN also exerted a promotory effect on lipid accumulation in HepG2 cells under normal glucose conditions (NG, 5 mM) and liver of normal fed zebrafish larvae. High glucose (HG, 25 mM)-induced lipid accumulation was suppressed by l-glutamine-d-fructose 6-phosphate amidotransferase inhibitors. ER stress inhibitors did not suppress HG or GlcN-mediated lipid accumulation. HG and GlcN stimulated protein expression, DNA binding and O-GlcNAcylation of carbohydrate-responsive element-binding protein (ChREBP). Furthermore, both HG and GlcN increased nuclear sterol regulatory element-binding protein-1 (SREBP-1) levels in HepG2 cells. In contrast to its stimulatory effect under NG, GlcN suppressed lipid accumulation in HepG2 cells under HG conditions. Similarly, GlcN suppressed lipid accumulation in livers of overfed zebrafish. In addition, GlcN activity on DNA binding and O-GlcNAcylation of ChREBP was stimulatory under NG and inhibitory under HG conditions. Moreover, GlcN enhanced ChREBP, SREBP-1c, ACC, FAS, L-PK and SCD-1 mRNA expression under NG but inhibited HG-induced upregulation in HepG2 cells. The O-GlcNAc transferase inhibitor, alloxan, reduced lipid accumulation by HG or GlcN while the O-GlcNAcase inhibitor, PUGNAc, enhanced lipid accumulation in HepG2 cells and liver of zebrafish larvae. GlcN-induced lipid accumulation was inhibited by the AMPK activator, AICAR. Phosphorylation of AMPK (p-AMPK) was suppressed by GlcN under NG while increased by GlcN under HG. PUGNAc downregulated p-AMPK while alloxan restored GlcN- or HG-induced p-AMPK inhibition. Our results collectively suggest that GlcN regulates lipogenesis by sensing the glucose or energy states of normal and excess fuel through AMPK modulation.     

Sirtuin 3 is essential for hypertension‐induced cardiac fibrosis via mediating pericyte transition

Hypertension is the key factor for the development of cardiac fibrosis and diastolic dysfunction. Our previous study showed that knockout of sirtuin 3 (SIRT3) resulted in diastolic dysfunction in mice. In the present study, we explored the role of SIRT3 in angiotensin II (Ang‐II)–induced cardiac fibrosis and pericyte‐myofibroblast transition. NG2 tracing reporter NG2‐DsRed mouse was crossed with wild‐type (WT) mice and SIRT3KO mice. Cardiac function, cardiac fibrosis and reactive oxygen species (ROS) were measured. Mice infused with Ang‐II for 28 days showed a significant reduction of SIRT3 expression in the mouse hearts. Knockout of SIRT3 sensitized Ang‐II‐induced elevation of isovolumic relaxation time (IVRT) and reduction of ejection fraction (EF) and fractional shortening (FS). Ang‐II‐induced cardiac fibrosis, capillary rarefaction and hypertrophy were further enhanced by knockout of SIRT3. NG2 pericyte tracing reporter mice infused with Ang‐II had a significantly increased number of NG2‐DsRed pericyte in the heart. Knockout of SIRT3 further enhanced Ang‐II‐induced increase of pericytes. To examine pericyte‐myofibroblast/fibroblast transition, DsRed pericytes were co‐stained with FSP‐1 and α‐SMA. Ang‐II infusion led to a significant increase in numbers of DsRed⁺/FSP‐1⁺ and DsRed⁺/α‐SMA⁺ cells, while SIRT3KO further developed pericyte‐myofibroblast/fibroblast transition. In addition, knockout of SIRT3 promoted Ang‐II‐induced NADPH oxidase‐derived ROS formation together with increased expression of transforming growth factor beta 1 (TGF‐β1). We concluded that Ang‐II induced cardiac fibrosis partly by the mechanisms involving SIRT3‐mediated pericyte‐myofibroblast/fibroblast transition and ROS‐TGF‐β1 pathway.     

脑出血大鼠血肿周围脑组织含水量与MMP-9、IL-6及TIMP-1表达水平的相关性研究

目的:研究脑出血(ICH)大鼠血肿周围脑组织含水量与基质金属蛋白酶-9(MMP-9)、白细胞介素-6(IL-6)及组织基质金属蛋白酶抑制剂-1(TIMP-1)表达水平的相关性。方法:84只健康成年雄性Wistar大鼠按随机数字表法分成观察组、假手术组以及对照组,每组28只。另将观察组分成ICH后1d亚组9只,3d亚组9只,7d亚组10只。观察组大鼠实施ICH模型的制备,假手术组的大鼠仅给予空针穿刺,对照组不进行模型制备。检测并对比各组血肿周围的脑组织含水量与MMP-9、IL-6、TIMP-1水平,对比观察组内不同亚组(ICH后1d、3d、7d)血肿周围的脑组织含水量、MMP-9、IL-6和TIMP-1水平,并分析ICH大鼠血肿周围的脑组织含水量与MMP-9、IL-6、TIMP-1表达水平的相关性。结果:观察组血肿周围的脑组织含水量与MMP-9、IL-6、TIMP-1水平均分别高于假手术组及对照组,差异均有统计学意义(P0.05)。观察组内3d和7d亚组血肿周围的脑组织含水量与MMP-9、IL-6、TIMP-1水平均分别高于1d亚组,但7d亚组低于3d亚组,差异均有统计学意义(P0.05)。根据Spearman相关性分析结果显示,ICH大鼠血肿周围的脑组织含水量与MMP-9、IL-6、TIMP-1表达水平均呈正相关(P0.05)。结论:ICH大鼠的血肿周围脑组织含水量与MMP-9、IL-6及TIMP-1表达水平均呈正相关,MMP-9、IL-6、TIMP-1在ICH后周围组织水肿的发生、发展中具有重要作用。     

TAT-mediated peroxiredoxin 5 and 6 protein transduction protects against high-glucose-induced cytotoxicity in retinal pericytes

AimsHyperglycemia-induced oxidative stress is implicated in pericyte apoptosis seen in diabetic retinopathy. The six mammalian Peroxiredoxins (PRDXs) comprise a novel family of antioxidative proteins that negatively regulate oxidative stress-induced apoptosis by controlling reactive oxygen species (ROS) levels.Main methodsSprague–Dawley rats were used to detect the retinal expressions of PRDXs1–6. Pig pericytes cultured in high-glucose medium were used to monitor the protective effect of PRDX5 and 6 against high-glucose-associated change. Recombinant PRDX5 and 6 proteins were linked to the Trans-Activating Transduction (TAT) domain from HIV-1 TAT protein for their efficient delivery into cells/tissues.Key findingsWe found higher expression of PRDX5 and 6 mRNAs and PRDX5 and 6 proteins in retina than the other Prdxs (Prdx1–4). Western blotting affirmed the intracellular presence of TAT-linked proteins and revealed the efficient transduction of TAT-HA-PRDX5 and 6 in these cells. Extrinsic supply of TAT-HA-PRDX5 and 6 proteins inhibited the oxidative stress-induced DNA damage after high-glucose exposure in pig pericytes. The cell survival and apoptosis assay revealed that extrinsic supply of TAT-HA-PRDX5 and 6 proteins was responsible for inhibiting hyperglycemia-induced pericyte apoptosis.SignificanceResults suggest that delivery of PRDX5 and 6 might protect hyperglycemia-induced pericyte loss to inhibit oxidative stress.     

Generation of reactive oxygen species in sperms of rats as an earlier marker for evaluating the toxicity of endocrine-disrupting chemicals

Abstract

Bisphenol A (BPA) and diethylstilbestrol (DES) have been reported to cause sperm toxicity. To identify an earlier marker of toxicity of environmental substances or food additives, this study determined whether the levels of reactive oxygen species (ROS) in sperms could serve as indices for the prediction of sperm toxicity and quality. Male Wistar rats were given drinking water containing various doses of BPA or DES for 8 weeks. Some rats were treated with 0.45% N-acetyl cysteine (NAC) for 2 days prior to the administration of DES or BPA. Administration of BPA or DES to rats for 1 week dose-dependently increased the production of ROS, even at doses and time points which had no effect on sperm motility. 4-Hydroxy-2-nonenal modified proteins increased in sperms 8 weeks after BPA or DES treatment. NAC reversed oxidative stress and prevented the loss of sperm function in the DES or BPA-treated group. During observation, changes in the sperm motility, sperm count and morphology were not correlated to the increase in ROS levels. These results suggest that ROS levels may be used as an early indicator of sperm count and quality decreases which result from chronic toxicity.     

Late transplantation of allogeneic bone marrow stromal cells improves neurologic deficits subsequent to intracerebral hemorrhage

Background aimsStem cell therapy seems to be a promising therapeutic tool for treating central nervous system (CNS) injuries. Bone marrow stromal cell (BMSC) transplantation influences functional outcome subsequent to intracerebral hemorrhage (ICH), and enhances endogenous neurogenesis in acute condition studies. We investigated whether late administration of BMSC improves functional deficits subsequent to ICH.MethodsExperimental ICH was induced by stereotactic injection of 0.5 IU collagenase type IV in the striatum of adult female Wistar rats, and 2 months later intralesional administration of 5 × 10⁶ allogeneic BMSC from male donors rats in saline (n = 10), or saline only (n = 10), was performed. In the following 6 months, functional outcome was evaluated in each animal by rotarod, modified neurologic severity score (mNSS) and video-tracking box (VTB) tests. To study the behavior of BMSC after transplantation, in situ hybridization studies were performed, with double labeling of the chromosome Y-linked SrY-gene, and neuronal nuclei (NeuN) protein or gliofibrillary acidic protein (GFAP).ResultsThe assessment test revealed significant improvements in functional outcome for the BMSC-treated animals after 2 months of follow-up. Histologic results showed that functional outcome was associated with strong reactivation of endogenous neurogenesis. Furthermore, intralesional BMSC not only integrated in the injured tissue but also showed phenotypic expression of GFAP and NeuN.ConclusionsLate intracerebral transplantation of allogeneic BMSC induces functional recovery after ICH. The possibility of using this type of cell therapy to reverse the consequences of hemorrhagic stroke in humans should be considered.     

Ghrelin治疗脑出血大鼠脑水肿及MMP-9表达的影响

目的:研究Ghrelin对大鼠脑出血后脑水肿及脑组织中基质金属蛋白酶-9(MMP-9)表达的影响。方法:选取雄性SD大鼠80只,随机分为对照组(NC组)20只、假手术组(SHAM组)20只、脑出血组(ICH组)20只、Ghrelin治疗组(Ghrelin组)20只。利用自体动脉血注入法建立大鼠脑出血模型;Ghrelin组于建立脑出血模型后经股静脉注射Ghrelin 10 nmol/Kg·d。分别于12 h、24 h、3d、5 d、7 d时间点根据Berderson评分法评估各组大鼠神经系统功能;利用干湿重法测定各组大鼠脑组织含水量;利用蛋白质免疫印迹法(WB)检测脑组织中MMP-9表达情况。结果:在12 h、24 h、3 d、5 d、7 d,ICH组、Ghrelin组大鼠Berderson评分及脑组织含水量高于NC组、SHAM组(P0.05);在5 d、7 d,ICH组大鼠Berderson评分及脑组织含水量高于Ghrelin组(P0.05)。WB结果表明在12 h、24 h、3 d、5 d、7 d,ICH组大鼠脑组织中MMP-9的表达均高于NC组、SHAM组(P0.05);Ghrelin组MMP-9的表达在12 h、24 h、3 d高于NC组、SHAM组(P0.05),在5 d、7 d,与NC组、SHAM组无明显差异(P0.05);在5 d、7 d,ICH组MMP-9表达高于Ghrelin组(P0.05)。结论:在本研究中,Ghrelin可以在5 d后降低脑出血大鼠脑组织中MMP-9的表达程度,从而减轻脑水肿,改善脑出血大鼠神经功能。     

Purinergic modulation of glucose uptake into cultured rat podocytes: effect of diabetic milieu

Extracellular purines act via P1 and P2 receptors on podocytes and may influence on their function. This action may be modified under various (patho)physiological conditions leading to development of podocytopathy. Aim of study was to investigate effects of diabetic milieu, represented by high glucose concentration (HG, 30 mM glucose) on purinergic-induced changes of 2-deoxy-d-glucose (2-DG) uptake and on extracellular purines metabolism in cultured rat podocytes. Basal 2-DG uptake was 2.7-fold enhanced in HG compared to normal glucose concentration, NG (1271 ± 86 vs. 477 ± 37 nmol/h/mg protein, P < 0.001). ATP stimulated 2-DG uptake by 44 ± 4% and 29 ± 5% in NG and HG, respectively. ATP analogues, β, γ-methylene ATP and 2-methylthio ATP stimulated 2-DG uptake in range of 18–34% in NG and 16–17% in HG. Benzoylbenzoyl ATP increased 2-DG uptake about 24 ± 2% in NG however, its effect in HG reached 50 ± 1%. The antagonists of P2 receptors (suramin, reactive blue 2, PPADS) decreased basal 2-DG uptake in NG and HG; suramin and reactive blue 2 at average of 15 ± 4% in NG but in HG the effect was in following order: suramin 28 ± 3%; PPADS 20 ± 3% and RB-2 9 ± 0.9%. Extracellular adenosine concentration was higher in HG than in NG (0.48 ± 0.01 vs. 5.05 ± 0.39 μM, P < 0.05), however intracellular ATP content and extracellular ATP concentration were not affected. Neither ecto-ATPase nor ecto-5′-nucleotidase activities were affected in HG. In conclusion, diabetic milieu affects purinergic modulation of glucose transport into podocytes which may play a role in development of diabetic podocytopathy.     

The Effect of Statin on Epithelial-Mesenchymal Transition in Peritoneal Mesothelial Cells

Background

Statins have recently been highlighted for their pleiotropic actions distinct from cholesterol-lowering effects. Despite this interest, it is currently unknown whether statin therapy inhibits peritoneal dialysis (PD)-related epithelial-mesenchymal transition (EMT).

Methods

In vitro, human peritoneal mesothelial cells (HPMCs) were exposed to 5.6 mM glucose (NG) or 100 mM glucose (HG) with or without simvastatin (1 µM). In vivo, PD catheters were inserted into 32 Sprague-Dawley rats, and saline (C, n = 16) or 4.25% peritoneal dialysis fluid (PDF) (PD, n = 16) was infused for 4 weeks. Eight rats from each group were treated with 5 mg/kg/day of simvastatin intraperitoneally. Changes in the protein expression of EMT markers such as E-cadherin, α-SMA, Snail, and fibronectin in HPMCs and the peritoneum were evaluated by Western blot analysis and immunofluorescence or immunohistochemical staining. We also explored whether activation of the mevalonate pathway and its downstream small GTPases were involved in dialysis-related peritoneal EMT and could be inhibited by statin treatment.

Results

Compared to NG cells, E-cadherin expression was significantly decreased, while α-SMA, Snail, and fibronectin expression were significantly increased in HPMCs exposed to HG, and these changes were abrogated by simvastatin (p<0.05). In addition, the cobblestone-like appearance of normal HPMCs was converted into a fibroblast-like morphology after HG treatment, which was reversed by simvastatin. These EMT-like changes were also observed in HPMCs treated with geranyl-geranyl pyrophosphate (5 µM). HG significantly increased the protein expression of RhoA and Rac1 in the membrane fractions, and these increases were ameliorated by simvastatin (p<0.05). In PD rats, E-cadherin in the peritoneum was significantly decreased, whereas α-SMA, Snail, and fibronectin expression were significantly increased (p<0.05) compared to C rats. The thickness of the mesothelial layer in the peritoneum were also significantly greater in PD rats than in C rats (p<0.05). These changes of the peritoneum in PD rats were significantly attenuated by simvastatin.

Conclusion

This study demonstrated that PD-related EMT was mediated via the mevalonate pathway, and statin treatment inhibited the EMT changes in HG-treated HPMCs and PDF-stimulated PD rats. These findings suggest that statins may be a promising therapeutic strategy for preservation of peritoneal membrane integrity in long-term PD patients.     

Increase of reactive oxygen species in different tissues during lipopolysaccharide-induced fever and antipyresis: an electron paramagnetic resonance study

Abstract

Fever is a regulated increase in body temperature and a component of the acute-phase response, triggered mainly after the invasion of pathogens in the body. Reactive oxygen species (ROS) are generated during the physiological and pathological processes, and can act as both signalling molecules as well as promoters of oxidative stress. Male Wistar rats, pretreated with oral doses of acetaminophen, celecoxib, dipyrone, or ibuprofen 30?min before an intravenous lipopolysaccharide (LPS) or sterile saline injection, showed a reduced febrile response in all animals tested. The formation of ROS in the fresh blood, liver, brown adipose tissue (BAT), and hypothalamus of febrile and antipyretic-treated animals was assessed by electron paramagnetic resonance using the spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH). While the CM^? concentrations remained unaltered in the blood samples examined 5?h after the induction of fever, we found increased CM^? levels in the liver (in µM, saline: 290?±?42; LPS: 512?±?34), BAT (in µM, saline: 509?±?79, LPS: 855?±?79), and hypothalamus (in µM, saline: 292?±?35; LPS: 467?±?8) at the same time point. Importantly, none of the antipyretics were seen to alter the CM^? accumulation profile. Data from this study suggest that there is an increased formation of ROS in the different tissues during fever, which may cause oxidative stress, and that the antipyretics tested do not interfere with ROS production.     

Zinc-aggravated M1 microglia regulate astrocytic engulfment via P2×7 receptors

BackgroundGlial cells such as astrocytes and microglia play an important role in the central nervous system via communication between these glial cells. Activated microglia can exhibit either the inflammatory M1 phenotype or the anti-inflammatory M2 phenotype, which influences astrocytic neuroprotective functions, including engulfment of cell debris. Recently, extracellular zinc has been shown to promote the inflammatory M1 phenotype in microglia through intracellular zinc accumulation and reactive oxygen species (ROS) generation.PurposeHere, we investigated whether the zinc-enhanced inflammatory M1 phenotype of microglia affects the astrocytic engulfing activity.MethodsEngulfing activity was assessed in astrocytes treated with microglial-conditioned medium (MCM) from lipopolysaccharide (LPS)-activated or from ZnCl₂-pretreated LPS-activated M1 microglia. The effect of zinc on microglia phenotype was also validated using the zinc chelator N,N,N’,N’-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and the ROS scavenger Trolox.ResultsAlthough treatment of astrocytes with LPS showed no significant effect on the engulfing activity, MCM from LPS-induced M1 microglia increased the beads uptake by astrocytes. This increased uptake activity was suppressed when MCM from LPS-induced M1 microglia pretreated with ZnCl₂ was applied to astrocytes, which was further abolished by the intracellular zinc chelator TPEN and the ROS scavenger Trolox. In addition, expression of P2×7 receptors (P2×7R) was increased in astrocytes treated with MCM derived from M1 microglia but not in the M1 microglia pretreated with ZnCl₂.ConclusionThese findings suggest that zinc pre-treatment abolishes the ability of LPS-induced M1 microglia to increase the engulfing activity in astrocytes via alteration of astrocytic P2×7R.     

C3a Receptor Antagonist Ameliorates Inflammatory and Fibrotic Signals in Type 2 Diabetic Nephropathy by Suppressing the Activation of TGF-β/smad3 and IKBα Pathway

Objective

Diabetic nephropathy (DN) is a serious complication for patients with diabetes mellitus (DM). Emerging evidence suggests that complement C3a is involved in the progression of DN. The aim of this study was to investigate the effect of C3a Receptor Agonist (C3aRA) on DN and its potential mechanism of action in rats with type 2 diabetes mellitus (T2DM).

Methods

T2DM was induced in SD rats by a high fat diet (HFD) plus repeated low dose streptozocin (STZ) injections. T2DM rats were treated with vehicle or C3aRA for 8 weeks. Biochemical analysis, HE and PAS stains were performed to evaluate the renal function and pathological changes. Human renal glomerular endothelial cells (HRGECs) were cultured and treated with normal glucose (NG), high glucose (HG), HG+C3a, HG+C3a+C3aRA and HG+C3a+BAY-11-7082 (p-IKBα Inhibitor) or SIS3 (Smad3 Inhibitor), respectively. Real-time PCR, immunofluorescent staining and western blot were performed to detect the mRNA and protein levels, respectively.

Results

T2DM rats showed worse renal morphology and impaired renal function compared with control rats, including elevated levels of serum creatinine (CREA), blood urea nitrogen (BUN) and urine albumin excretion (UACR), as well as increased levels of C3a, C3aR, IL-6, p-IKBα, collagen I, TGF-β and p-Smad3 in the kidney of T2DM rats and C3a-treated HRGECs. In contrast, C3aRA treatment improved renal function and morphology, reduced CREA, UACR and the intensity of PAS and collagen I staining in the kidney of T2DM rats, and decreased C3a, p-IKBα, IL-6, TGF-β, p-Smad3 and collagen I expressions in HRGECs and T2DM rats.

Conclusion

C3a mediated pro-inflammatory and pro-fibrotic responses and aggravated renal injury in T2DM rats. C3aRA ameliorated T2DN by inhibiting IKBα phosphorylation and cytokine release, and also TGF-β/Smad3 signaling and ECM deposition. Therefore, complement C3a receptor is a potential therapeutic target for DN.