Rb1 postconditioning attenuates liver warm ischemia-reperfusion injury through ROS-NO-HIF pathway

AimsGinsenoside Rb1 could prevent ischemic neuronal death and focal cerebral ischemia, but its roles to liver warm I/R injury remain to be defined. We determined if Rb1 would attenuate warm I/R injury in mice.Main methodsMice were divided into sham, I/R, Rb1 + I/R (Rb1 postconditioning, 20 mg/kg, i.p. after ischemia), sham + L-NAME, I/R + L-NAME, and Rb1 + I/R + L-NAME groups using 60 min of the liver median and left lateral lobes ischemia. Serum levels of alanine aminotransferase (ALT) were measured and morphology changes of livers were evaluated. Contents of nitric oxide (NO) and nitric oxide synthase (NOS), malondialdehye (MDA) and activity of superoxide dismutase (SOD) were measured. Expressions of Akt, p-Akt, iNOS, HIF-1alpha, tumor necrosis factor-a (TNF-α) and intercellular adhesion molecule-1 (ICAM-1) were also determined by western blot or immunohistochemistry.Key findingsRb1 postconditioning attenuated the dramatically functional and morphological injuries. The levels of ALT were significantly reduced in Rb1 group (p < 0.05). Rb1 upregulated the concentrations of NO, iNOS in serum, iNOS, and activity of SOD in hepatic tissues (p < 0.05), while it dramatically reduced the concentration of MDA (p < 0.05). Protein expressions of p-Akt, iNOS and HIF-1alpha were markedly enhanced in Rb1 group. Protein and mRNA expressions of TNF-α and ICAM-1 were markedly suppressed by Rb1 (p < 0.05).SignificanceWe found that Rb1 postconditioning could protect liver from I/R injury by upregulating the content of NO and NOS, and also HIF-1alpha protein expression. These protective effects could be abolished by L-NAME. These findings suggested Rb1 may have the therapeutic potential through ROS-NO-HIF pathway for management of liver warm I/R injury.     

Dipeptidyl peptidase-IV inhibition prevents blood–retinal barrier breakdown,inflammation and neuronal cell death in the retina of type 1 diabetic rats

Diabetic retinopathy, a leading cause of vision loss in working-age population, is often associated with inflammation and apoptosis. We have previously reported that sitagliptin, a DPP-IV inhibitor, exerts beneficial effects in the retina of type 2 diabetic animals. The present study aimed to evaluate whether sitagliptin can exert protective effects in the retina of type 1 diabetic animals by a mechanism independent of insulin secretion and glycemia normalization.Streptozotocin-induced diabetic rats were treated orally with sitagliptin (5 mg/kg/day) for the last two weeks of 4 weeks of diabetes. Sitagliptin treatment did not change the weight and glucose, HbA_1c or insulin levels. However, it prevented the diabetes-induced increase in DPP-IV/CD26 activity and levels in serum and retina. Sitagliptin also prevented the increase in blood–retinal barrier (BRB) permeability and inhibited the changes in immunoreactivity and endothelial subcellular distribution of occludin, claudin-5 and ZO-1 proteins induced by diabetes. Furthermore, sitagliptin decreased the retinal inflammatory state and neuronal apoptosis.Sitagliptin inhibited the BRB breakdown in a type 1 diabetic animal model, by a mechanism independent of normalization of glycemia, by preventing changes in tight junctions (TJs) organization. Sitagliptin also exerted protective effects against inflammation and pro-apoptotic state in the retina of diabetic rats. Altogether, these results suggest that sitagliptin might be envisaged to be used to prevent or delay some of the alterations associated with the development of diabetic retinopathy.     

Role of high-fat diet in regulation of gene expression of drug metabolizing enzymes and transporters

AimsLate phase ischemic preconditioning (LPC) protects the heart against ischemia–reperfusion (I/R) injury. However, its effect on myocardial tissue oxygenation and related mechanism(s) is unknown. The aim of the current study is to determine whether LPC attenuates post-ischemic myocardial tissue hyperoxygenation through preserving mitochondrial oxygen metabolism.Main methodsC57BL/6 mice were subjected to 30 min coronary ligation followed by 60 min or 24 h reperfusion with or without LPC (3 cycles of 5 min I/5 min R): Sham, LPC, I/R, and LPC + I/R group. Myocardial tissue Po₂ and redox status were measured with electron paramagnetic resonance (EPR) spectroscopy.Key findingsUpon reperfusion, tissue Po₂ rose significantly above the pre-ischemic level in the I/R mice (23.1 ± 2.2 vs. 12.6 ± 1.3 mm Hg, p < 0.01). This hyperoxygenation was attenuated by LPC in the LPC + I/R mice (11.9 ± 2.0 mm Hg, p < 0.01). Activities of NADH dehydrogenase (NADH-DH), succinate-cytochrome c reductase (SCR) and cytochrome c oxidase (CcO) were preserved or increased in the LPC group, significantly reduced in the I/R group, and conserved in the LPC + I/R group. Manganese superoxide dismutase (Mn-SOD) protein expression was increased by LPC in the LPC and LPC + I/R mice compared to that in the Sham control (1.24 ± 0.01 and 1.23 ± 0.01, p < 0.05). Tissue redox status was shifted to the oxidizing state with I/R (0.0268 ± 0.0016/min) and was corrected by LPC in the LPC + I/R mice (0.0379 ± 0.0023/min). Finally, LPC reduced the infarct size in the LPC + I/R mice (10.5 ± 0.4% vs. 33.3 ± 0.6%, p < 0.05).SignificanceThus, LPC preserved mitochondrial oxygen metabolism, attenuated post-ischemic myocardial tissue hyperoxygenation, and reduced I/R injury.     

Inhibition of semicarbazide-sensitive amine oxidase attenuates myocardial ischemia-reperfusion injury in an in vivo rat model

AimsThis study tested the hypothesis that the inhibition of semicarbazide-sensitive amine oxidase (SSAO) after ischemia could attenuate myocardial ischemia–reperfusion (I/R) injury.Main methodsAnesthetized male Sprague–Dawley rats underwent myocardial I/R injury. Saline, semicarbazide (SCZ, 30 mg/kg), hydralazine (HYD, 10 mg/kg), or LJP 1207 (30 mg/kg) was administered intraperitoneally 3 min before reperfusion. After 30 min of ischemia and 180 min of reperfusion, the myocardial infarct size was determined using nitroblue tetrazolium staining. Myocardial myeloperoxidase activity was determined through biochemical assay. HE staining was used for histopathological evaluation. Myocardial SSAO activity was assayed with high performance liquid chromatography analysis. Additionally, the endothelial expression of P-selectin was evaluated using immunohistochemistry after 30 min of ischemia and 20 min of reperfusion.Key findingsMyocardial SSAO activity was increased in myocardial I/R injury. Administration of SCZ, HYD, or LJP 1207 reduced the myocardial infarct size and decreased leukocyte infiltration and endothelial P-selectin expression in myocardial I/R injury in vivo.SignificanceThese data suggest that myocardial I/R injury up-regulates myocardial SSAO activity, and the inhibition of SSAO prior to reperfusion is able to attenuate acute myocardial I/R injury.     

Effects of Canarium odontophyllum leaves on plasma glucose and T lymphocyte population in streptozotocin-induced diabetic rats

Type 1 diabetes mellitus is a chronic disease characterized by lack of insulin production. Immune mechanisms are implicated in the pathogenesis of Type 1 diabetes. Canarium odontophyllum (CO) fruits and leaves have been shown to possess high antioxidant activity. This study was conducted to evaluate the effects of CO leaves aqueous extract on the blood glucose and T lymphocyte population in the spleen of streptozotocin (STZ)-induced diabetic rats. Nineteen male Sprague–Dawley rats were randomly divided into three groups: normal, diabetic control and CO treated diabetic groups. Diabetes was induced by a single intraperitoneal injection of 65 mg STZ/kg body weight. The extract of CO leaves was administered orally by force feeding daily at the dose of 300 mg/kg for 28 days. The rats were sacrificed at the end of the study and the spleen was harvested for flow cytometry analysis. The results showed a significant decrease in body weight of diabetic and CO treated diabetic groups compared with the normal group (p < 0.05). The fasting blood glucose level of CO treated diabetic group was significantly lower than the diabetic group (p < 0.05). Diabetic and CO treated diabetic groups showed a significant increase in the percentage of spleen CD3⁺ CD4⁺ T lymphocytes (p < 0.05) when compared with the normal group. However, there was no significant difference in the percentage of spleen CD3⁺ CD8⁺ T lymphocytes among all experimental groups. The finding suggested that an aqueous extract of CO leaves has the ability to reduce blood glucose levels in diabetic rats.     

Renal GLUT1 reduction depends on angiotensin-converting enzyme inhibition in diabetic hypertensive rats

AimsAngiotensin-converting enzyme (ACE) inhibitors are used in diabetic kidney disease to reduce systemic/intra-glomerular pressure. The objective of this study was to investigate whether reducing blood pressure (BP) could modulate renal glucose transporter expression, and urinary markers of diabetic nephropathy in diabetic hypertensive rats treated with ramipril or amlodipine.Main methodsDiabetes was induced in spontaneously-hypertensive rats (~ 210 g) by streptozotocin (50 mg/kg). Thirty days later, animals received ramipril 15 μg/kg/day (R, n = 10), or amlodipine 10 mg/kg/day (A, n = 8,) or water (C, n = 10) by gavage. After 30-day treatment, body weight, glycaemia, urinary albumin and TGF-β1 (enzyme-linked immunosorbent assay) and BP (tail-cuff pressure method) were evaluated. Kidneys were removed for evaluation of renal cortex glucose transporters (Western blotting) and renal tissue ACE activity (fluorometric assay).Key findingsAfter treatments, body weight (p = 0.77) and glycaemia (p = 0.22) were similar among the groups. Systolic BP was similarly reduced (p < 0.001) in A and R vs. C (172.4 ± 3.2; 186.7 ± 3.7 and 202.2 ± 4.3 mm Hg; respectively). ACE activity (C: 0.903 ± 0.086; A: 0.654 ± 0.025, and R: 0.389 ± 0.057 mU/mg), albuminuria (C: 264.8 ± 15.4; A: 140.8 ± 13.5 and R: 102.8 ± 6.7 mg/24 h), and renal cortex GLUT1 content (C: 46.81 ± 4.54; A: 40.30 ± 5.39 and R: 26.89 ± 0.79 AU) decreased only in R (p < 0.001, p < 0.05 and p < 0.001; respectively).SignificanceWe concluded that the blockade of the renin–angiotensin system with ramipril reduced early markers of diabetic nephropathy, a phenomenon that cannot be specifically related to decreased BP levels.     

Different roles of zinc plus arachidonic acid on insulin sensitivity between high fructose- and high fat-fed rats

AimsThis study was to determine the effects of zinc plus arachidonic acid (ZA) treatment on the insulin action in the specific ZA target organs using hyperinsulinemic euglycemic clamp method.Main methods18 Sprague–Dawley rats weighing ~ 130 g were divided into 3 groups of 6 rats and treated them with 1) normal rat chow, 2) high fructose (60.0%) diet only, or 3) the same fructose diet plus drinking water containing 10 mg zinc plus 50 mg arachidonic acid (AA)/L. In a separate study, male Wistar rats weighing ~ 250 g were fed normal rat chow (n = 4) or high fat (66.5%) diet with drinking water containing zero (n = 9) or 10 mg AA plus 20 mg zinc /L (n = 9). After 4 week treatment, insulin action was assessed using the hyperinsulinemic eguglycemic clamp technique.Key findingsHigh fructose feeding impaired suppression of hepatic glucose output by insulin compared to controls during the clamp procedure (4.39 vs. 2.35 mg/kg/min; p < 0.05). However, ZA treatment in high fructose-fed rats showed a significant improvement of hepatic insulin sensitivity compared to non-treatment controls (4.39 vs. 2.18 mg/kg/min; p < 0.05). Glucose infusion rates in Wistar rats maintained on a high fat diet (HFD) were significantly lower compared to control rats (22.8 ± 1.3 vs. 31.9 ± 1.4 mg/kg/min; p < 0.05). ZA treatment significantly improved (~ 43%) peripheral tissue insulin sensitivity in HFD fed animals (26.7 ± 1.3 [n = 9] vs. 22.8 ± 1.3 mg/kg/min; p < 0.05).SignificanceThese data demonstrate that ZA treatment is effective in improving glucose utilization in hyperglycemic rats receiving either a high-fructose or a high-fat diet.     

17-β estradiol in the acetylcholinesterase activity and lipid peroxidation in the brain and blood of ovariectomized adult and middle-aged rats

AimsTo investigate the 17-β estradiol in the acetylcholinesterase activity and lipid peroxidation in the brain and blood of ovariectomized rats of different ages.Main methodsAnimals were randomly assigned into three experimental groups of each age (n = 6). Control groups consisted of adult (sham-A) and middle-aged (sham-MA) female rats, ovariectomized adult (OVX-A) and middle-aged (OVX-MA) rats without estrogen therapy reposition, and ovariectomized adult (OVX + E2-A) and middle-aged (OVX + E2-MA) rats treated with 17-β estradiol for 30 days. After this period, AChE activity and lipid peroxidation were measured in the brain and blood.Key findingsThe AChE activity increased (p < 0.05) in striatum (ST) in OVX-A, OVX + E2-A and OVX-MA, and hippocampus (HP) in OVX-MA. The enzyme activity decreased (p < 0.05) in ST of OVX + E2-MA, and cerebral cortex (CC) in OVX + E2-A, OVX-MA and OVX + E2-MA. Blood AChE activity increased (p < 0.05) in OVX + E2-A and decreased (p < 0.05) in OVX-MA. Lymphocyte AChE activity increased (p < 0.05) in OVX-A and OVX + E2-A and decreased (p < 0.05) in OVX-MA. Lipid peroxidation increased (p < 0.05) in ST of OVX-A, CC of OVX-A and OVX-MA, HP of OVX-A, and cerebellum (CE) of OVX-A, OVX-MA, and OVX + E2-MA. Lipid peroxidation decreased (p < 0.05) in ST, CC and CE of OVX + E2-A, and ST and HP of OVX + E2-MA. Similar values of lipid peroxidation to control groups were found in ST and HP of OVX-MA, HP of OVX + E2-A and CC of OVX + E2-MA.Significance17-β estradiol is able to modulate the AChE activity and non-neuronal cholinergic response as well as to reduce lipid peroxidation. Its response is dependent on the age and brain structure analyzed.     

3-Bromo-7-nitroindazole attenuates brain ischemic injury in diabetic stroke via inhibition of endoplasmic reticulum stress pathway involving CHOP

AimsThe role of nitric oxide (NO) and endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of cerebral ischemic/reperfusion (I/R) injury and diabetes. The aim of the study was to investigate the neuroprotective potential of 3-bromo-7-nitroindazole (3-BNI), a potent and selective neuronal nitric oxide synthase (nNOS) inhibitor against ER stress and focal cerebral I/R injury associated with comorbid type 2 diabetes in-vivo.Main methodsType 2 diabetes was induced by feeding high-fat diet and streptozotocin (35 mg/kg) treatment in rats. Focal cerebral ischemia was induced by 2 h middle cerebral artery occlusion (MCAO) followed by 22 h of reperfusion. Immunohistochemistry and western blotting methods were employed for the detection and expression of ER stress/apoptosis markers [78 kDa glucose regulated protein (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP)]. TUNEL assay for DNA fragmentation was also performed.Key findingsThe diabetic rats subjected to cerebral I/R had prominent neurological damage and functional deficits compared with sham-operated rats. Massive DNA fragmentation was observed in ischemic penumbral region of diabetic brains. Concomitantly, the enhanced immunoreactivity and expression of ER stress/apoptosis markers were noticed. 3-BNI (30 mg/kg, i.p.) treatment significantly inhibited the cerebral infarct, edema volume and improved functional recovery of neurological deficits. The neuroprotection was further evident by lesser DNA fragmentation with a concomitant reduction of GRP78 and CHOP.SignificanceThe study demonstrates the neuroprotective potential of 3-BNI in diabetic stroke model which may be partly due to inhibition of ER stress pathway involving CHOP.     

Increased secretion of insulin and proliferation of islet β-cells in rats with mesenteric lymph duct ligation

Background &; aimsIt has been suggested that intestinal lymph flow plays an important role in insulin secretion and glucose metabolism after meals. In this study, we investigated the influence of ligation of the mesenteric lymph duct on glucose metabolism and islet β-cells in rats.MethodsMale Sprague–Dawley rats (10 weeks old) were divided into two groups: one underwent ligation of the mesenteric lymph duct above the cistern (ligation group), and the other underwent a sham operation (sham group). After 1 and 2 weeks, fasting plasma concentrations of glucose, insulin, triglyceride, glucose-dependent insulinotropic polypeptide (GIP), and the active form of glucagon-like peptide-1 (GLP-1) were measured. At 2 weeks after the operation, the oral glucose tolerance test (OGTT) and intravenous glucose tolerance test (IVGTT) were performed. After the rats had been sacrificed, the insulin content of the pancreas was measured and the proliferation of β-cells was assessed immunohistochemically using antibodies against insulin and Ki-67.ResultsDuring the OGTT, the ligation group showed a significant decrease in the plasma glucose concentration at 120 min (p < 0.05) and a significant increase in the plasma insulin concentration by more than 2-fold at 15 min (p < 0.01). On the other hand, the plasma GIP concentration was significantly decreased at 60 min (p < 0.01) in the ligated group, while the active form of GLP-1 showed a significantly higher level at 90 min (1.7-fold; p < 0.05) and 120 min (2.5-fold; p < 0.01). During the IVGTT, the plasma insulin concentration in the ligation group was significantly higher at 2 min (more than 1.4-fold; p < 0.05). Immunohistochemistry showed that the ratios of β-cell area/acinar cell area and β-cell area/islet area, and also β-cell proliferation, were significantly higher in the ligation group than in the sham group (p < 0.05, p < 0.01 and p < 0.01, respectively). The insulin content per unit wet weight of pancreas was also significantly increased in the ligation group (p < 0.05).ConclusionsIn rats with ligation of the mesenteric lymph duct, insulin secretion during the OGTT or IVGTT was higher, and the insulin content and β-cell proliferation in the pancreas were also increased. Our data show that mesenteric lymph duct flow has a role in glucose metabolism.     

Protective outcomes of low-dose doxycycline on renal function of Wistar rats subjected to acute ischemia/reperfusion injury

Renal ischemia-reperfusion injury (IRI) is a major cause of acute renal failure. Doxycycline (Dc) belongs to the tetracycline-class of antibiotics with demonstrated beneficial molecular effects in the brain and heart, mainly through matrix metalloproteinases inhibition (MMP). However, Dc protection of renal function has not been demonstrated. We determined whether low doses of Dc would prevent decreases in glomerular filtration rate (GFR) and maintain tubular Na⁺ handling in Wistar rats subjected to kidney I/R. Male Wistar rats underwent bilateral kidney ischemia for 30 min followed by 24 h reperfusion (I/R). Doxycycline (1, 3, and 10 mg/kg, i.p.) was administered 2 h before surgery. Untreated I/R rats showed a 250% increase in urine volume and proteinuria, a 60% reduction in GFR, accumulation of urea-nitrogen in the blood, and a 60% decrease in the fractional Na⁺ excretion due to unbalanced Na⁺ transporter activity. Treatment with Dc 3 mg/kg maintained control levels of urine volume, proteinuria, GFR, blood urea-nitrogen, fractional Na⁺ excretion, and equilibrated Na⁺ transporter activities. The Dc protection effects on renal function were associated with kidney structure preservation and prevention of TGFβ and fibronectin deposition. In vitro, total MMP activity was augmented in I/R and inhibited by 25 and 50 μM Dc. In vivo, I/R augmented MMP-2 and -9 protein content without changing their activities. Doxycycline treatment downregulated total MMP activity and MMP-2 and -9 protein content. Our results suggest that treatment with low dose Dc protects from IRI, thereby preserving kidney function.     

Protective effect of heme oxygenase-1 induction against hepatic injury in alcoholic steatotic liver exposed to cold ischemia/reperfusion

AimsThe purpose of this study was to investigate the cytoprotective role of heme oxygenase-1 (HO-1) induction in hepatic injury in alcoholic steatotic liver exposed to cold ischemia/reperfusion (I/R).Main methodsAnimals were fed an ethanol liquid diet or isocaloric control diet for 5 weeks. Isolated perfused rat livers were preserved in Histidine–Tryptophan–Ketoglutarate at 4 °C. After 24 h of storage, livers were subjected to 120 min of reperfusion with Krebs–Henseleit bicarbonate buffer at 37 °C. Animals were pretreated with cobalt protoporphyrin (CoPP, 5 mg/kg, i.p.) or zinc protoporphyrin (ZnPP, 25 mg/kg, i.p.), HO-1 inducer and antagonist, respectively.Key findingsIn the model of ischemia/isolated perfusion, endogenous HO-1 was downregulated in the livers fed with ethanol diet (ED I/R). In ED I/R group, portal pressure and lactate dehydrogenase release were significantly increased, while bile output and hyaluronic acid clearance decreased compared to rats fed on control diet (CD I/R). Furthermore, hepatic glutathione content decreased and lipid peroxidation increased in the ED I/R group compared to the CD I/R group. These alterations were attenuated by upregulation of HO-1 with CoPP pretreatment.SignificanceOur results suggest that chronic ethanol consumption aggravates hepatic injury during cold I/R and it is likely due to downregulation of endogenous HO-1. Prior induction of HO-1 expression may provide a new strategy to protect livers against hepatic I/R injury or to increase the donor transplant pool through modulation of marginal alcoholic steatotic livers.     

The characteristics of therapeutic effect of pinocembrin in transient global brain ischemia/reperfusion rats

AimsThe therapeutic effect of pinocembrin, together with the therapeutic time window, in the transient global cerebral ischemia/reperfusion (I/R) rats was investigated.Main methodsAdult male Sprague–Dawley rats were subjected to global cerebral ischemia for 20 min by four-vessel occlusion. Pinocembrin (1 and 5 mg/kg) was administrated intravenously 30 min before ischemia and 30 min, 2 h, 6 h after reperfusion, respectively. Neurological scores, brain edema and histological examination by Nissl staining were employed to assess the neuronal injury after ischemia and the neuroprotection by pinocembrin. The activities of superoxide dismutase (SOD), myeloperoxidase (MPO) and the content of malondialdehyde (MDA) in brain tissue were tested by colorimetric assays. Alterations of neurotransmitters were determined by a high performance liquid chromatography–electrochemical method.Key findingsPinocembrin significantly ameliorated neurological deficits and brain edema, and alleviated the degree of hippocampal neuronal loss at 24 h after global cerebral I/R with a broad therapeutic time window. It was found that treatment with pinocembrin reduced the compensatory increase of SOD activity and decreased the MDA level and MPO activity in a dose-dependent manner. The metabolic balance between excitatory and inhibitory amino acids was modulated by pinocembrin treatment.SignificanceThese findings suggest that pinocembrin provides neuroprotection against global cerebral ischemic injury with a wide therapeutic time window, which may be attributed to its antioxidative, antiinflammatory and antiexcitotoxic effects.     

Dragon's blood dropping pills have protective effects on focal cerebral ischemia rats model

Dragon's blood is a bright red resin obtained from Dracaena cochinchinensis (Lour.) S.C.Chen (Yunnan, China). As a traditional Chinese medicinal herb, it has great traditional medicinal value and is used for wound healing and to stop bleeding. Its main biological activity comes from phenolic compounds. In this study, phenolic compounds were made into dropping pills and their protective effects were examined by establishing focal cerebral ischemia rats model used method of Middle Cerebral Artery Occlusion (MCAO), and by investigating indexes of neurological scores, infarct volume, cerebral index, cerebral water content and oxidation stress. Compared to model group, high, middle and low groups of Dragon's blood dropping pills could improve the neurological function significantly (p < 0.01) and reduce cerebral infarct volume of focal cerebral ischemia rats remarkably (p < 0.05–0.01). Meanwhile, each group could alleviate cerebral water content and cerebral index (p < 0.05–0.01) and regulate oxidative stress of focal cerebral ischemia rats obviously (p < 0.05–0.01). Activities of middle group corresponded with that treated with positive control drug. The results obtained here showed that Dragon's blood dropping pills had protective effects on focal cerebral ischemia rats.     

Baicalin upregulates the genetic expression of antioxidant enzymes in Type-2 diabetic Goto-Kakizaki rats

AimThe primary purpose of this study was to characterize and investigate the antioxidant and anti-diabetic activities of the flavonoid baicalin in type 2 diabetic Goto-Kakizaki rats.Main methodsFour groups of Goto-Kakizaki rats (n = 6) were subjected to the following oral treatments for 30 days: (1) metformin — 500 mg/kg (2) baicalin — 120 mg/kg (3) metformin 500 mg/kg and baicalin — 120 mg/kg (4) vehicle treated diabetic controls receiving distilled water. The plasma glucose, triglyceride, total cholesterol, lipid peroxide and protein carbonyl contents were measured on a weekly basis. Following the completion of the treatment, the rats were sacrificed and their blood, heart, pancreatic and hepatic tissues were collected for analysis. The antioxidant enzyme activities as well as their expression were quantified using Western Blot, microarray and RT-PCR.Key findingsThe respective analyses showed that the baicalin- and the metformin and baicalin-treated groups had statistically significant increases (p < 0.05) in the activity and expression of the antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) compared with vehicle- and metformin-treated groups. Further complementing the antioxidant enzyme activity increases, the oxidative stress markers of plasma lipid peroxide and protein carbonyl contents were reduced in these groups as well. These treatment groups also had reduced plasma total cholesterol and triglyceride levels compared with vehicle-treated and metformin-treated groups (p < 0.05).SignificanceBaicalin was an efficient antioxidant in reducing hyperglycemia-induced oxidative stress through the increased expression of antioxidant enzyme activities. It was also an efficient anti-hypertriglyceridemic as well as anti-hypercholesterolemic agent compared with metformin.     

Placental antiangiogenic prolactin fragments are increased in human and rat maternal diabetes

Introduction/objectivesThe role of the placenta in diabetic mothers on fetal development and programming is unknown. Prolactin (PRL) produced by decidual endometrial cells may have an impact. Although full-length PRL is angiogenic, the processed form by bone morphogenetic protein-1 (BMP-1) and/or cathepsin D (CTSD) is antiangiogenic.The objectives were to investigate the involvement of decidual PRL and its antiangiogenic fragments in placentas from type-1 diabetic women (T1D) and from pregnant diabetic rats with lower offspring weights than controls.MethodsPRL, BMP-1, and CTSD gene expressions and PRL protein level were assessed in T1D placentas (n = 8) at delivery and compared to controls (n = 5). Wistar rats received, at day 7 of pregnancy, streptozotocin (STZ) (n = 5) or nicotinamide (NCT) plus STZ (n = 9) or vehicle (n = 9). Placental whole-genome gene expression and PRL western blots were performed at birth.ResultsIn human placentas, PRL (p < 0.05) and BMP-1 (p < 0.01) gene expressions were increased with a higher amount of cleaved PRL (p < 0.05) in T1D than controls. In rats, diabetes was more pronounced in STZ than in NCT–STZ group with intra-uterine growth restriction. Decidual prolactin-related protein (Dprp) (p < 0.01) and Bmp-1 (p < 0.001) genes were up-regulated in both diabetic groups, with an increased cleaved PRL amount in the STZ (p < 0.05) and NCT–STZ (p < 0.05) groups compared to controls. No difference in CTSD gene expression was observed in rats or women.ConclusionsAlterations in the levels of the PRL family are associated with maternal diabetes in both rats and T1D women suggesting that placental changes in these hormones impact on fetal development.     

Aclidinium bromide, a novel long-acting muscarinic antagonist for COPD with improved preclinical renal and urinary safety profile

AimsAclidinium bromide is a novel, long-acting, inhaled muscarinic antagonist currently in registration phase for the treatment of chronic obstructive pulmonary disease. Since urinary difficulty and retention have been reported for anticholinergic agents such as tiotropium and ipratropium, it is important to examine the preclinical urinary and renal safety profile of aclidinium.Main methodsThe effect of aclidinium on urine and electrolyte excretion, renal function and voiding cystometry was analysed in conscious water-loaded Wistar rats (10–1000 μg/kg, s.c.), anaesthetised Beagle dogs (1000 μg/kg, i.v.) and anaesthetised guinea pigs (3–100 μg/kg, intratracheally), respectively. Aclidinium plasma levels were determined in an independent study. Active comparators were tiotropium (all studies) and ipratropium (cystometry only).Key findingsAclidinium 1000 μg/kg had no effect on urine excretion in rats, in contrast to tiotropium 100 μg/kg which significantly decreased this parameter (p < 0.05). Aclidinium 1000 μg/kg also had no effect on renal function in Beagle dogs. In guinea pigs, aclidinium 3–100 μg/kg had no effect on urinary bladder function, whereas tiotropium and ipratropium 100 μg/kg decreased the peak micturition pressure (p < 0.05), increased the volume of urine retained in the bladder (p < 0.01) and showed a trend to decrease the volume of urine excreted.SignificanceAclidinium had no significant effect on urinary and renal function in the animal models studied. These results, together with the rapid plasma clearance of aclidinium reported previously, suggest a lower propensity to induce urinary retention in humans than tiotropium and ipratropium.     

Hepatoprotective and antioxidant activity of N-Trisaccharide in different experimental rats

ObjectivesTo investigate the hepatoprotective, antioxidant and antihyperlipidemic effect of N-Trisaccharide isolated from Cucumis prophetarum (L.) on different experimental rats.MethodsN-Trisaccharide (25 and 50 mg/kg.b.w), silymarin (25 mg/kg) and glibenclamide (25 mg/kg) was orally administered once daily for 28 days and toxicity evaluation studies were carried out. Liver damage was assessed by determining DNA damage, serum enzyme activities and hepatic histopathology of carbon tetrachloride (CCl₄) induced hepatic injury in rats. Enzymatic and non enzymatic antioxidant levels in liver and kidney were determined and biochemical parameters such as, serum lipid profile, renal function markers were estimated in type 2 diabetic rats.ResultsDNA fragmentation analysis revealed the protective effect of N-Trisaccharide on liver DNA damage. Histopathological studies indicated that CCl₄-induced liver injury was less severe in N-Trisaccharide (25 and 50 mg/kg) treated group. Given at the above doses conferred significant protection against the hepatotoxic actions of CCl₄ in rats, reducing serum markers like SGOT, SGPT, ALP, creatinine and urea levels back to near normal (p < 0.05) compared to untreated rats. In diabetic rats, N-Trisaccharide treatment significantly reversed abnormal status of enzymatic and non-enzymatic antioxidants levels to near normal. Also, serum lipids such as TG, TC, LDL-C and VLDL-C levels were significantly (p < 0.05) reduced compared to diabetic untreated rats.ConclusionPresent study results confirm that N-Trisaccharide possesses significant antihyperlipidemic, antioxidant and hepatoprotective properties.     

The receptor for advanced glycation endproducts and its ligands in patients with myasthenia gravis

ObjectiveMyasthenia gravis (MG) is a T- and B-cell mediated autoimmune disorder affecting the neuromuscular junction. The receptor for advanced glycation endproducts (RAGE) plays a role in the amplification of chronic inflammatory disorders and autoimmune diseases. We sought to investigate the role of RAGE and its ligands in the pathophysiology of MG.MethodsIn this cross-sectional study we enrolled 42 patients with MG and 36 volunteers. We employed enzyme-linked immunosorbent assays to determine the concentration of soluble RAGE (sRAGE) and high mobility group box 1 (HMGB1) in serum of patients and volunteers. In a subpopulation of patients we measured the serum levels of endogenous secretory (es) RAGE and various RAGE ligands, such as S100B, S100A8 and advanced glycation endproducts (AGE-CML). Reported are means and standard error mean.ResultsWe found significantly reduced levels of the soluble receptors sRAGE and esRAGE in patients with MG compared to volunteers without MG (sRAGE [pg/ml] 927.2 ± 80.8 vs. 1400.1 ± 92.4; p < 0.001; esRAGE [pg/ml] 273.5 ± 24.6 vs. 449.0 ± 22.4; p < 0.001). Further categorization of patients with MG according to the distribution of muscle involvement revealed the following sRAGE concentrations: generalized MG 999.4 ± 90.8 and ocular MG 696.1 ± 161.8 (vs. control; One-way ANOVA: p < 0.001; Post hoc analysis: generalized vs. ocular MG: p = 0.264, generalized MG vs. control: p = 0.008, ocular MG vs. control: p = 0.001). In patients with detectable antibodies specific for acetylcholine receptors (Anti-AChR positive) the sRAGE concentration was 970.0 ± 90.2 compared to those without (seronegative) 670.6 ± 133.1 (vs. control; One-way ANOVA: p < 0.001; Post hoc analysis: Pos vs. Neg.: p = 0.418, Pos vs. control: p = 0.003, Neg. vs. control: p = 0.008). We next investigated the role of RAGE ligands in MG. The concentrations of RAGE ligands in patients with MG and controls were as follows: (HMGB1 [ng/ml] 1.7 ± 0.1 vs. 2.1 ± 0.2; p = 0.058; S100B [pg/ml] 22.5 ± 22.5 vs. 14.4 ± 9.2; p = 0.698; S100A8 [pg/ml] 107.0 ± 59.3 vs. 242.5 ± 103.6; p = 0.347; and AGE-CML [ng/ml] 1100.8 ± 175.1 vs. 1399.8 ± 132.8; p = 0.179).ConclusionsOur data suggest a role for the RAGE pathway in the pathophysiology of MG. Further studies are warranted to elucidate more about this immunological axis in patients with MG.     

Activation of glucose transport during simulated ischemia in H9c2 cardiac myoblasts is mediated by protein kinase C isoforms

Glucose transport into cells may be regulated by a variety of conditions, including ischemia. We investigated whether some enzymes frequently involved in the metabolic adaptation to ischemia are also required for glucose transport activation. Ischemia was simulated by incubating during 3 h H9c2 cardiomyoblasts in a serum- and glucose-free medium in hypoxia. Under these conditions 2-deoxy-d-[2,6-³H]-glucose uptake was increased (57% above control levels, p < 0.0001) consistently with GLUT1 and GLUT4 translocation to sarcolemma. Tyrosine kinases inhibition via tyrphostin had no effect on glucose transport up-regulation induced by simulated ischemia. On the other hand, chelerythrine, a broad range inhibitor of protein kinase C isoforms, and rottlerin, an inhibitor of protein kinase C delta, completely prevented the stimulation of the transport rate. A lower activation of hexose uptake (19%, p < 0.001) followed also treatment with Gö6976, an inhibitor of conventional protein kinases C. Finally, PD98059-mediated inhibition of the phosphorylation of ERK 1/2, a downstream mitogen-activated protein kinase (MAPK), only partially reduced the activation of glucose transport induced by simulated ischemia (31%, p < 0.01), while SB203580, an inhibitor of p38 MAPK, did not exert any effect. These results indicate that stimulation of protein kinase C delta is strongly related to the up-regulation of glucose transport induced by simulated ischemia in cultured cardiomyoblasts and that conventional protein kinases C and ERK 1/2 are partially involved in the signalling pathways mediating this process.