Ya-Ping Deng

Glibenclamide attenuates myocardial injury by lipopolysaccharides in streptozotocin-induced diabetic mice

BackgroundSepsis is a common disease that continues to increase in incidence in the world. Diseases, such as diabetes mellitus, may make the situation worse. Diabetic patients are at increased risk for common infections. This study was designed to investigate the role of glibenclamide on myocardial injury by lipopolysaccharides (LPS) in streptozotocin induced diabetic mice (STZ-mice).MethodsLPS was used to induce endotoxemia in STZ-mice. Heart rate and mean arterial pressure were measured by MPA-HBBS. Serum epinephrine level was measured by enzyme-linked immunosorbent assays (ELISA). Myocardial injury was examined by light and transmission electron microscope and TUNEL staining. Macrophage infiltration was measured by immunohistochemistry. Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) levels in myocardial tissue and serum in STZ-mice, and in conditional medium of primary cultured peritoneal macrophages were determined by ELISA. Nalp3 and Caspase-1 protein levels were measured by Western blotting analysis.ResultsSTZ administration decreased body weight and increased blood glucose in C57BL/6 mice. LPS injection caused decreases of heart rate and mean arterial pressure, and elevated serum epinephrine level in C57BL/6 mice. Compared with control mice without STZ treatment, LPS induced more severe myocardial injury and macrophage infiltration in STZ-mice, which was attenuated by pretreatment of glibenclamide. LPS stimulation enhanced the levels of IL-1β and TNF-α in both cardiac tissue and serum. Glibenclamide pretreatment significantly inhibited the serum levels of pro-inflammatory cytokines. Either high glucose or LPS increased the levels of IL-1β and TNF-α in the conditional medium of peritoneal macrophages. Glibenclamide treatment suppressed the increase of IL-1β level induced by high glucose and LPS. Furthermore, Nalp3 and Caspase-1 levels were markedly increased by high glucose plus LPS, and both proteins were significantly inhibited by glibenclamide treatment.ConclusionsWe conclude that glibenclamide could attenuate myocardial injury induced by LPS challenge in STZ-mice, which was possibly related to inhibiting inflammation through Nalp3 inflammasomes.

Simvastatin attenuates radiation-induced tissue damage in mice

The aim of this study was to investigate the protective effect of simvastatin against radiation-induced tissue injury in mice. Mice were radiated with 4 Gy or 8 Gy after 20 mg/kg/d simvastatin treatment over 2 weeks. Morphological changes were observed in the jejunum and bone marrow, and apoptotic cells were determined in both tissues. Peripheral blood cells were counted, and the superoxide dismutase (SOD) activity and the malondialdehyde (MDA) level in tissues of both thymus and spleen were measured. Compared with the radiation-only group, 20 mg/kg/d simvastatin administration significantly increased the mean villi height and decreased apoptotic cells in jejunum tissue, and stimulated regeneration and reduced apoptotic cells in bone marrow. Peripheral blood cell analysis revealed that simvastatin treatment induced a larger number of red blood cells and increased the hemoglobin level present after 4 Gy of radiation. Interestingly, it was also found that the number of peripheral endothelial progenitor cells was markedly increased following simvastatin administration. Antioxidant determination for tissues displayed that simvastatin therapy increased the SOD activity after both 4 and 8 Gy of radiation, but only decreased the MDA level after 4 Gy. Simvastatin ameliorated radiation-induced tissue damage in mice. The radioprotective effect of simvastatin was possibly related to inhibition of apoptosis and improvement of oxygen-carrying and antioxidant activities.

Establishment of Tube Formation Assay of Bone Marrow-Derived Endothelial Progenitor Cells

Stroke is the second most common cause of death and a leading cause of adult disability worldwide [1,2]. Endothelial dysfunction has been observed in stroke patients and has been related to stroke physiopathology, clinical severity, and outcome [3]. Endothelial progenitor cells (EPCs) are bone marrow-derived cells that are mobilized to the peripheral circulation when vascular repair and neovascularization are required. EPCs play a critical role in maintaining endothelial function and might affect the progression of vascular disease [4]. Assays that measure EPC number and function may provide important information for the risk stratification of stroke. It has been demonstrated that the tube formation assay is a robust, rapid, reproducible, and comprehensive method [5]. However, bone marrow (BM)-EPCs’ ability to form tubular-like network remains controversial. The overwhelming majority of the works assessed the tube formation function of BM-EPCs by coplating with mature endothelial cells, such as human umbilical vein endothelial cells (HUVECs) [6,7]. In this study, we established a BM-EPCs tube formation method, which is able to generate tubular-like network on Matrigel without coplating with mature endothelial cells. Mouse bone marrow-derived EPCs were isolated and cultured according to described technique [8]. Cultured BM-EPCs were characterized as cells coexpressing Sca-1 and Flk-1 (BD Pharmingen, San Diego, CA, USA) under flow cytometry examination. To confirm the BM-EPCs phenotypes, cells were stained for the uptake of Dil-acLDL (Molecular Probes Inc., Eugene, OR, USA) and FITC-labeled Ulex europaeus agglutinin (lectin; Sigma-Aldrich, St. Louis, MO, USA). The in vitro angiogenic activity of BM-EPCs was determined by Matrigel tube formation assay. Briefly, BMEPCs were replated at the density of 30,000, 60,000, 120,000, and 180,000 cells per cm (10,000, 20,000, 40,000 and 60,000 cells per well) in 96-well plates precoated with 50 ll/well growth factor-reduced Matrigel (BD Biosciences, Bedford, MA, USA). Tube formation ability of BM-EPCs was assessed by tubes number. Tube was defined as a structure exhibiting a length 4 times its width [9]. Values are presented as mean SD. Multiple comparisons involving more than three groups were performed using one-way ANOVA. Post hoc comparisons were performed with the Neuman–Keuls test. A value of P < 0.05 was considered statistically significant. Mouse BM-EPCs grew to confluence and displayed a typical spindle-like morphology (Figure 1A). Flow cytometric analysis showed about 17.3% of cell population was positive for Flk-1 and Sca-1 (Figure 1B). Characterization of BM-EPCs was further confirmed as Dil-acLDL and lectin double-positive adherent cells under a fluorescence microscope (Figure 1C). Examples of BM-EPCs tube formation on top of the gelled Matrigel are presented in Figure 2. Successful development of the dispersed BM-EPCs to tubular-like network mainly depends on both the culture time and the cell density on the Matrigel. Plated BM-EPCs initially attached on the Matrigel, then migrated toward each other over the next 2–4 h, and gradually formed tubular-like network, which matured by 6–9 h. After that, the network was prone to detach from the Matrigel and break apart (Figure 2A). Cell density is another critical determinant. Inadequate cell density yielded incomplete networks, while too many cells resulted in

Role of Kir6.2 subunits of ATP-sensitive potassium channels in endotoxemia-induced cardiac dysfunction

BackgroundCardiac dysfunction is well-described in endotoxemia and diagnosed in up to 60% of patients with endotoxic shock. ATP-sensitive potassium (KATP) channels are critical to cardiac function. This study investigates the role of Kir6.2 subunits of KATP channels on cardiac dysfunction in lipopolysaccharide (LPS)-induced endotoxemia.MethodsKir6.2 subunits knockout (Kir6.2−/−) and wild-type (WT) mice were injected with LPS to induce endotoxemia. Cardiac function was monitored by echocardiography. Left ventricles were taken for microscopy (both light and electron) and TUNEL examination. Serum lactate dehydrogenase (LDH) and creatine kinase (CK) activities, and tumor necrosis factor-α (TNF-α) levels in both serum and left ventricular tissues were determined.ResultsCompared to WT, Kir6.2−/− mice showed significantly declined cardiac function 360 min after LPS administration, aggravated myocardial damage and elevated serum LDH and CK activities. Apoptotic cells were obviously increased in heart tissues from Kir6.2−/− mice at 90, 180 and 360 min. TNF-α expression in both serum and heart tissues of Kir6.2−/− mice was significantly increased.ConclusionsWe conclude that Kir6.2 subunits are critical in resistance to endotoxemia-induced cardiac dysfunction through reducing myocardial damage by inhibition of apoptosis and inflammation. KATP channels blockers are extensively used in the treatment of diabetes, their potential role should therefore be considered in the clinic when patients treated with antidiabetic sulfonylureas are complicated by endotoxemia.

Metformin ameliorates insulitis in STZ-induced diabetic mice

Background & Aims Metformin is currently the most widely used first-line hypoglycemic agent for diabetes mellitus. Besides glucose-lowering action, there is increasingly interest in the potential anti-inflammatory action of this drug. In the present study, we investigated the actions of metformin on experimental insulitis using STZ-induced diabetic mice. Methods Mice with acute diabetes induced by STZ were administered metformin by gavage. Changes of blood glucose and body weight, and the daily amount of food and water intake were measured. Pancreatic tissues were collected for histologic analyses. Pathological assessment and immunohistochemistry analysis were used to determine the effect of metformin on insulitis. Inflammatory cytokines in the pancreas and insulin levels were measured through ELISA analysis. Results Metformin significantly reduced blood glucose levels and improved aberrant water intake behavior in experimental diabetic mice. No significant differences were observed in terms of body weight and food intake behavior in metformin-treated animals. In the STZ-induced model of diabetes, we found the appearance of pronounced insulitis. However, metformin administration reduced the severity of insulitis assessed by blind pathological scoring. In addition, metformin treatment improved insulin levels in experimental diabetic mice. ELISA assay revealed decreased levels of inflammatory response marker IL-1β and TNF-α in the pancreatic tissues following metformin treatment. Conclusion Metformin attenuated insulitis in the STZ-induced mice model of diabetes. This islet-protective effect might be partly correlated with the anti-inflammatory action of metformin.

PPARα Agonist Stimulated Angiogenesis by Improving Endothelial Precursor Cell Function Via a NLRP3 Inflammasome Pathway

Background: Impaired wound healing is a common complication of diabetes and is the leading cause of lower extremity amputation. Treatment with fenofibrate, a peroxisome proliferators–activated receptor α (PPARα) agonist, was associated with a lower risk of amputations, particularly minor amputations without known large-vessel diseases, probably through non-lipid mechanisms. The current study aimed to test our hypothesis that fenofibrate stimulates angiogenesis and restores endothelial precursor cell (EPC) function via inhibiting Nod-like receptor protein 3 (NLRP3) inflammasome in streptozotocin (STZ)-induced diabetic mice. Methods: Male C57BL/6 mice were randomly divided into three groups: control, STZ-induced diabetic mice and fenofibrate treated diabetic group. Wound closure was assessed by wound area and CD31 positive capillaries. Both the migration and tube formation capacities of EPCs were measured. Intracellular nitric oxide (NO) and superoxide (O2-) levels were determined. Activity of NLRP3 inflammasome in EPCs was assessed by measuring thioredoxin-interacting protein (TXNIP), NLRP3, and caspase-1 expression. Results: Compared with the untreated diabetic mice, wound closure and capillary densities were significantly increased in fenofibrate treated group. Fenofibrate treatment restored EPC function, increased NO production, and decreased O2- level in EPCs of diabetic mice. Furthermore, fenofibrate deregulated the activity of NLRP3 inflammasome by reducing TXNIP, NLRP3 and caspase-1 expression in EPCs of diabetic mice. In vitro, fenofibrate prevented high glucose induced EPC dysfunction, deregulated NLRP3 inflammasome activity. In addition, fenofibrate inhibited IL-1β expression caused by combination use of high glucose and lipopolysaccharide. Conclusion: Fenofibrate can accelerate wound healing in diabetic mice, which at least in part was mediated by improving the impaired EPC function via a NLRP3 inflammasome pathway, suggesting the significance of PPARα agonists in the treatment of diabetes.

Acarbose Accelerates Wound Healing via Akt/eNOS Signaling in db/db Mice

Refractory wound is a dreaded complication of diabetes and is highly correlated with EPC dysfunction caused by hyperglycemia. Acarbose is a widely used oral glucose-lowering drug exclusively for T2DM. Previous studies have suggested the beneficial effect of acarbose on improving endothelial dysfunction in patients with T2DM. However, no data have been reported on the beneficial efficacy of acarbose in wound healing impairment caused by diabetes. We herein investigated whether acarbose could improve wound healing in T2DM db/db mice and the possible mechanisms involved. Acarbose hastened wound healing and enhanced angiogenesis, accompanied by increased circulating EPC number in db/db mice. In vitro, a reversed BM-EPC dysfunction was observed after the administration of acarbose in db/db mice, as reflected by tube formation assay. In addition, a significantly increased NO production was also witnessed in BM-EPCs from acarbose treated db/db mice, with decreased O2 levels. Akt inhibitor could abolish the beneficial effect of acarbose on high glucose induced EPC dysfunction in vitro, accompanied by reduced eNOS activation. Acarbose displayed potential effect in promoting wound healing and improving angiogenesis in T2DM mice, which was possibly related to the Akt/eNOS signaling pathway.

Metformin accelerates wound healing in type 2 diabetic db/db mice

Wound healing impairment is increasingly recognized to be a consequence of hyperglycemia-induced dysfunction of endothelial precursor cells (EPCs) in type 2 diabetes mellitus (T2DM). Metformin exhibits potential for the improvement of endothelial function and the wound healing process. However, the underlying mechanisms for the observed beneficial effects of metformin application remain to be completely understood. The present study assessed whether metformin, a widely used therapeutic drug for T2DM, may accelerate wound closure in T2DM db/db mice. Genetically hyperglycemic db/db mice were used as the T2DM model. Metformin (250 mg/kg/day; intragastric) was administered for two weeks prior to EPC collection and wound model creation in db/db mice. Wound healing was evaluated by alterations in the wound area and the number of platelet endothelial cell adhesion molecule-positive cells. The function of the isolated bone marrow-derived EPCs (BM-EPCs) was assessed by a tube formation assay. The number of circulating EPCs, and the levels of intracellular nitric oxide (NO) and superoxide (O2−) were detected by flow cytometry. Thrombospondin-1 (TSP-1) expression was determined by western blot analysis. It was observed that treatment with metformin accelerated wound healing, improved angiogenesis and increased the circulating EPC number in db/db mice. In vitro, treatment with metformin reversed the impaired BM-EPC function reflected by tube formation, and significantly increased NO production while decreasing O2− levels in BM-EPCs from db/db mice. In addition, TSP-1 expression was markedly attenuated by treatment with metformin in cultured BM-EPCs. Metformin contributed to wound healing and improved angiogenesis in T2DM mice, which was, in part, associated with stimulation of NO, and inhibition of O2− and TSP-1 in EPCs from db/db mice.

Cognitive Function is Impaired by Obesity and Alleviated by Lorcaserin Treatment in Mice

Alzheimer’s disease (AD), a progressive neurodegenerative disease, is the most common type of dementia in the elderly population. Much effort has been investigated in the risk factors of early onset of AD. Accumulating evidences have shown that western sedentary lifestyle may lead to cognitive dysfunction [1]. Childhood and adult obesity is increasing dramatically and obese individuals who show early impairment of cognitive performance could have earlier onset of dementia. As for the treatment of obesity, diet and exercise have been the mainstays of weight management in the obese population, but with limited efficacy [2]. Thus, other treatment modalities and medications might be required for some patients. Orlistat, an inhibitor of pancreatic lipases, has been the only remaining antiobesity drug till June 2012 when lorcaserin (a selective 5-HT2C receptor agonist) was approved by the FDA. We initiated this study to determine whether cognitive dysfunction was linked to obesity and anti-obesity medications. Diet-induced obese mouse model was established using 40% high fat diet as previously described [3]. We adopted Morris water maze (MWM) to evaluate spatial learning and memory in C57BL/ 6 mice. Oxidative stress in the mouse hippocampus was assessed by dihydroethidium (DHE) fluorescent staining. Function to adhesion/migrate of bone marrow endothelial progenitor cells (EPCs) and endothelium-dependent vasodilation of thoracic aortae was evaluated to examine endothelial function in mice [4]. Obese mice exhibited impaired cognitive function in mice, which could be partially restored by lorcaserin or orlistat treatment. Latency of obese mice at the 5th training day was significantly longer than that of control mice; lorcaserin/orlistat significantly shortened the latency in anti-obesity treated obese mice (Figure 1A). When the hidden platform was removed, mice in obese group spent less time and distance in the original target quadrant compared with the control group; Lorcaserin/orlistat treatment significantly increased percentage of time and distance in the target quadrant in obese mice (Figure 2B). Furthermore, DHE staining assay showed that lorcaserin/orlistat treatment alleviated obesity-exacerbated oxidative stress in the hippocampus of C57BL/6 mice. We also found that dietary obesity significantly impaired EPCs function and the endothelium-dependent vasodilation of thoracic artery, which could be restored by lorcaserin/orlistat. Lorcaserin/ orlistat significantly ameliorated obesity-impaired BM-EPCs capacity to adhere (control: 1.00 0.054, HFD: 0.805 0.043, HFD + orlistat: 1.14 0.102, HFD + lorcaserin: 1.23 0.078; P < 0.01, Figure 2A) and migrate (control: 1.00 0.072, HFD: 0.538 0.051, HFD + orlistat: 1.13 0.121, HFD + lorcaserin: 1.38 0.102, P < 0.01, Figure 2B). Endothelium-dependent vasodilation of mouse artery was significantly impaired by obesity and restored by lorcaserin/orlistat administration (P < 0.01; Figure 2C). Obese individuals could have decreased life expectancy and increased cardiovascular risks including diabetes, hypertension, coronary artery disease, strokes, and even cancers. Recent studies showed that high-fat diet intake interferes with hippocampal functioning [5]. While studies in humans suggest that obesity is associated with disruptions in cognitive ability, results from animal studies were a little confusing, with some reports describing significant effects of obesity on performance in the Morris maze and others failing to detect effects of obesity on spatial learning. In the basis of previous studies, we conducted this study. It is shown that lorcaserin significantly alleviated obesity-impaired spatial learning/memory ability in DIO mice. This finding might be