Role of fibrogenic markers in chronic hepatitis C and associated hepatocellular carcinoma

Detection and follow up of fibrogenesis in chronic hepatitis C (CHC) is mandatory for early treatment and risk stratification. The current study included 120 patients with CHC, of whom 30 had liver cirrhosis (LC) and 30 had hepatocellular carcinoma (HCC). 15 wedge liver biopsies, taken during laparoscopic cholecystectomy, were included as normal controls. Cases were subjected to laboratory investigations, serologic markers for viral hepatitis and assessment of circulating levels of hyaluronic acid (HA) and platelet-derived growth factor (PDGF). Immunohistochemical expression of connective tissue growth factor (CTGF), PDGF and transforming growth factor-β1 (TGF-β1) was also carried out. A significant increase (p < 0.01) in serum HA was noticed in CHC, LC and HCC compared to controls. Although, a significant decrease in serum PDGF was detected in CHC and LC compared to controls, HCC values were comparable. A significant up-regulation of CTGF was detected in CHC, LC and HCC (p < 0.01) in contrast to its limited mild expression in normal livers. Intense PDGF positive staining was noticed in CHC, LC and HCC compared to scattered faint expression in controls. The significant expression and marked intensity of PDGF staining matched the progress to tumorigenesis. A positive TGF-β1 immunostaining was also noticed in CHC, LC and HCC. An intense and extensive cytoplasmic expression of TGF-β1 was encountered in patients with LC revealing that CTGF, PDGF and TGF-β1 act synergistically in LC. Data revealed that HA and CTGF may be implicated as important diagnostic parameters for assessment of hepatic fibrosis and PDGF for monitoring malignant transformation in CHC.     

−509C>T polymorphism in the TGF-β1 gene promoter,impact on the hepatocellular carcinoma risk in Chinese patients with chronic hepatitis B virus infection

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. The risk for developing HCC increases with severity of inflammation and fibrosis. Transforming growth factor-β1 (TGF-β1) is most frequently upregulated in tumor cells. The most studied −509C>T polymorphism of TGF-β1 gene has been associated with colorectal, gynecologic, and lung cancers. To assess whether this polymorphism in TGF-β1 gene is associated with susceptibility to and/or clinicopathologic characteristics of HBV-related HCC, a total of 575 patients with chronic HBV infection and 299 healthy volunteers with no evidence of recent or remote HBV infection were prospectively enrolled. The patients were divided into two groups: those without (n = 196) and those with HCC (n = 379). These 379 HCC patients with chronic HBV infection were designated as cases, the remaining 196 patients without HCC and 299 healthy volunteers served as disease and healthy controls, respectively. −509C>T polymorphism in the TGF-β1 gene promoter was studied using restriction fragment-length polymorphism. In addition, tumor tissues of liver (n = 60) were obtained from the studied HCC patients for measurement of TGF-β1 mRNA expression levels. We also assessed the plasma TGF-β1 levels of HBV patients without (n = 94) or with HCC (n = 136) and healthy subjects (n = 120). In our study group, the risk of HCC in Chinese patients with HBV infection was significantly lower with the TT genotypes than in those with the CC genotypes at position −509 of TGF-β1 gene (P = 0.01). In addition, in the case group, patients with the CC genotype had a statistically significant higher median plasma TGF-β1 or liver tumor tissue TGF-β1 mRNA level compared with the individuals with the TT genotype. However, in a subsequent analysis of the association between this polymorphism and clinicopathological characteristics including tumor number, size, grade, stage, and invasiveness, there was no significant difference in both the distribution of genotype or allelic frequency within HCC patients, indicating that −509C>T exchange in TGF-β1 gene may play an important role in the occurrence, not the progression of HBV-related HCC through influencing plasma concentrations of TGF-β1 or TGF-β1 mRNA expression of liver tumor tissue.     

Expression of connective tissue growth factor and interleukin-11 in intratumoral tissue is associated with poor survival after curative resection of hepatocellular carcinoma

In the present study, we evaluated the prognostic value of intratumoral and peritumoral expression of connective tissue growth factor (CTGF), transforming growth factor-beta 1 (TGF-β1), and interleukin-11 (IL-11) in patients with hepatocellular carcinoma (HCC) after curative resection. Expression of CTGF, TGF-β1, and IL-11 was assessed by immunohistochemical staining of tissue microarrays containing paired tumor and peritumoral liver tissue from 290 patients who had undergone hepatectomy for histologically proven HCC. The prognostic value of these and other clinicopathologic factors were evaluated. The median follow-up time was 54.3 months (range, 4.3–118.3 months). High intratumoral CTGF expression was associated with vascular invasion (P = 0.015), intratumoral IL-11 expression correlated with higher tumor node metastasis (TNM) stage (P = 0.009), and peritumoral CTGF overexpression correlated with lack of tumor encapsulation (P = 0.031). Correlation analysis of these proteins revealed that intratumoral CTGF and IL-11 correlated with high intratumoral TGF-β1 expression (r = 0.325, P < 0.001; and r = 0.273, P < 0.001, respectively). TNM stage (P < 0.001), high intratumoral CTGF levels (P = 0.010), and intratumoral IL-11 expression (P = 0.015) were independent prognostic factors for progression-free survival (PFS). Vascular invasion (P = 0.032), TNM stage (P < 0.001), high intratumoral CTGF levels (P = 0.036), and intratumoral IL-11 expression (P = 0.013) were independent prognostic factors for overall survival (OS). High intratumoral CTGF and intratumoral IL-11 expression were associated with PFS and OS after hepatectomy, and the combination of intratumoral CTGF with IL-11 may be predictive of survival.     

Downregulation of Apoptosis and Modulation of TGF-β1 by Sodium Selenate Prevents Streptozotocin-Induced Diabetic Rat Renal Impairment

To investigate whether sodium selenate treatment would impact on the onset of diabetic nephropathy, we examined blood glucose, serum biochemical components, and interrelationship between oxidative stress, TGF-β1, and apoptosis in streptozotocin (STZ) induced diabetic rats. Sixty male Wistar rats were divided into six groups. Group I (n = 10), normal control; Group II (n = 10), diabetic control; Group III (n = 10), sodium selenate (16 μmoles/kg) + diabetic; Group IV (n = 10), sodium selenate (32 μmoles/kg) + diabetic; Group V (n = 10), sodium selenate (16 μmoles/kg) control; and Group VI (n = 10), sodium selenate (32 μmoles/kg) control. Sodium selenate was administered via orogastric route for 10 weeks. In the diabetic group, diabetes was induced by single intraperitoneal injection of STZ (50 mg/kg). The levels of blood glucose were estimated and total cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides, creatinine, urea, and albumin were detected in serum. Antioxidant status was examined by measuring the superoxide dismutase (SOD), catalase, glutathione, and lipid peroxidation in kidney tissues. Histopathological studies were performed in the kidney tissue sections. The expression of TGF-β1 was estimated by the immunohistochemical analysis in kidneys. Apoptotic study in kidney was performed using the TdT-mediated dUTP nick end labeling technique. It was observed that blood glucose, serum, total cholesterol, HDL cholesterol, triglycerides, creatinine, urea, and albumin were significantly higher in diabetic control groups. Diabetic + sodium selenate (16 and 32 μmoles/kg) significantly reduced blood glucose, serum, total cholesterol, HDL cholesterol, triglycerides, creatinine, urea, and albumin levels. Selenium-treated groups significantly increased antioxidant enzyme activities (SOD, catalase, and glutathione) in kidneys of diabetic rats. All enzyme activities of selenium control groups did not differ compared with the normal control. Sodium selenate reduces significantly lipid peroxidation in diabetic rats. Cellular architecture of the diabetic rats was altered whereas sodium selenate administration rectifies the degenerative changes of the kidney. Profound immunopositivity of TGF-β1 was observed in the glomerular and tubulointerstitial cells of diabetic rat kidney. Immunopositivity of TGF-β1 was significantly reduced in both low and high dose of sodium-selenate-treated rats (P < 0.05, P < 0.01). High numbers of apoptotic cells were observed in diabetic rats whereas sodium selenate in both doses significantly reduces the incidence of apoptosis (P < 0.05, P < 0.01). We conclude herein that sodium selenate has the potential to play a significant role in limiting the renal impairment by altering the apoptosis and TGF-β1 in experimental diabetic rats.     

Growth inhibition induced by transforming growth factor-β1 in human oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is a world-wide health problem and its incidence accounts for 1.9–3.5% of all malignant tumors. Transforming growth factor beta/Smads (TGF-β/Smads) signaling pathway plays an important role in oncogenesis, but its function and molecular mechanisms in OSCC remain unclear. Expression of transforming growth factor-β receptor type II (TβRII) and Smad4 was studied by immunohistochemistry in 108 OSCC patients and 10 normal controls. Function and molecular mechanisms of TGF-β/Smads signaling pathway was then investigated in two human tongue squamous carcinoma cell lines with high and low metastasis (Tb and Tca8113) by RT-PCR, Western Blot, immunofluorescence, cell growth curve and flow cytometry (FCM), respectively. TβRII and Smad4 were significantly down-regulated in tumor tissues (with or without lymph node metastasis) compared to normal oral epithelium tissues (P < 0.05). TGF-β1 induced arrest of the cell cycle rather than cell death in Tca8113 and Tb cells, and this influence was mediated by the increasing the expression and changing the location of its downstream components of TGF-β1/Smads signaling pathway. TGF-β1 rapidly increased the expression of p15 and p21 in both Tca8113 and Tb cells. TGF-β1 did not increase p27 expression in Tca8113 cells, but p27 expression was increased in Tb cells. These indicated that TGF-β1 induced G₁ arrest of cell cycle through a different regulating pathway in Tb cells compared with Tca8113 cells. Thus, we conclude that TGF-β/Smads signaling pathway play a important role on cell growth and metastasis potential in OSCC. Xiumei Wang, Wenjing Sun, and Jing Bai contributed equally to this paper.     

Decreased α1-Adrenergic Receptor Binding in the Cerebral Cortex and Brain Stem during Pancreatic Regeneration in Rats

The purpose of this study was to investigate the role of brain α₁-adrenergic receptor binding in the rat model of pancreatic regeneration using 60–70% pancreatectomy. The α₁-adrenergic receptors kinetics was studied in the cerebral cortex and brain stem of sham operated, 72 h pancreatectomised and 7 days pancreatectomised rats. Scatchard analysis with [³H]prazosin in cerebral cortex and brain stem showed a significant decrease (P < 0.01), (P < 0.05) in maximal binding (B _max) with a significant decrease (P < 0.001), (P < 0.01) in the K _d in 72 h pancreatectomised rats compared with sham respectively. Competition analysis in cerebral cortex and brain stem showed a shift in affinity during pancreatic regeneration. The sympathetic activity was decreased as indicated by the significantly decreased norepinephrine level in the plasma (P < 0.001), cerebral cortex (P < 0.01) and brain stem (P < 0.001) of 72 h pancreatectomised rats compared to sham. Thus, from our results it is suggested that the central α₁-adrenergic receptors have a functional role in the pancreatic regeneration mediated through the sympathetic pathway.     

Effects of castration on aggression and levels of serum sex hormones and their central receptors in mandarin voles (Microtus mandarinus)

Aggression in socially monogamous mandarin vole (Microtus mandarinus) was observed after castration. Levels of serum sex hormones and their central receptors were also measured using enzyme-linked immunosorbent assay and immunohistochemistry methods. The data indicate that adult males showed higher levels of aggression after castration. However, castration significantly reduced levels of serum testosterone, and the number of androgen receptor immunoreactive neurons in the anterior hypothalamus, bed nucleus of the stria terminalis, medial amygdaloid nucleus (P < 0.01) and lateral septal nucleus (P < 0.05). In addition, levels of estrogen receptor β in the anterior hypothalamus and medial amygdaloid nucleus (P < 0.05), bed nucleus of the stria terminalis and lateral septal nucleus (P < 0.01) declined to varying degrees at weekly intervals. In contrast, serum 17β-estradiol concentrations were up-regulated by castration and castration did not change levels of estrogen receptor α in the medial amygdaloid nucleus and lateral septal nucleus, but increased it in the anterior hypothalamus 3 weeks after castration (P < 0.05). We suggest that higher levels of aggression induced by castration may be independent of serum testosterone and androgen receptors, and may be associated with high serum 17β-estradiol concentrations, stable estrogen receptor α immunoreactive neurons in some brain regions and the relative ratio of the two estrogen receptors.     

TGF-β1 reverses PDGF-stimulated migration of human aortic smooth muscle cells in vitro

Summary Platelet-derived growth factor (PDGF) and transforming growth factor beta-1(TGF-β1) were tested separately or together for the ability to stimulate migration of human aortic vascular smooth muscle cells (VSMC). PDGF (10 ng/ml) stimulated migration of VSMC over a 48-h period. TGF-β1 (10 ng/ml) had no effect on migration during the same period. VSMC exposed simultaneously to both TGF-β1 and PDGF exhibited diminished migration (50%) when compared to cells treated only with PDGF. Cells that migrated in the presence of PDGF possessed short actin cables that extended from cellular processes at the leading edge of migrating cells; focal adhesions containing the α_vβ₃/β₅ integrins localized to the same region. Cells grown in the presence of TGF-β1 exhibited long, intensely stained actin filaments that spanned the entire length of the cell and were similar to untreated control VSMC. Focal adhesions containing α_vβ₃/β₅ distributed evenly on the basal surface in both TGF-β1-treated cells and control cultures. Cellular responses to PDGF were mitigated when TGF-β1 was present in the culture medium. VSMC grown in the presence of both PDGF and TGF-β1 exhibited elongated actin filaments that were similar to nonmotile TGF-β1-treated cultures. Concomitant exposure of VSMC to PDGF and TGF-β1 resulted in focal adhesions that distributed evenly on the lower cell surface. This study suggests that TGF-β1 can partially reverse the stimulatory effect of PDGF on VSMC migration in vitro by modifying the actin cytoskeleton and the distribution of the α _vβ₃/β₅ integrins.     

Upregulation of BACE1 and β-Amyloid Protein Mediated by Chronic Cerebral Hypoperfusion Contributes to Cognitive Impairment and Pathogenesis of Alzheimer’s Disease

Chronic cerebral hypoperfusion (CCH) increases the risk of Alzheimer disease (AD) through several biologically plausible pathways, but the relationship between CCH and the development of AD remains uncertain. To investigate expression of APP, BACE1 and Aβ in the hippocampus of BCCAO rats and study pathophysiological mechanism of AD from CCH. CCH rat model was established by chronic bilateral common carotid artery occlusion (BCCAO). Behavior was evaluated after BCCAO with Morris water maze and open-field task. Expression of Aβ was measured by enzyme linked immunosorbent assay (ELISA). β-Amyloid precursor protein cleavage enzyme 1 (BACE1) and β-amyloid precursor protein (APP) were tested by ELISA, Western blotting and RT-PCR. Cognitive impairment occurred with CCH by Morris water maze test and open-field task. The BACE1 and Aβ level in BCCAO rats was more increased than sham-operation control rats (P < 0.01) but APP had no difference(P > 0.05). The expression of BACE1 and Aβ has no inter-grouop difference in BCCAO rats (P > 0.05). The level of BACE1 and Aβ had positive correlation with cognitive impairment (P < 0.01) while no correlation was observed between APP and cognitive impairment. Chronic cerebral ischemia contributes to cognitive impairment and vascular pathogenesis of Alzheimer’s disease that chronic cerebral hypoperfusion increases BACE1 and Aβ level in brain.     

Investigation of Serum Trace Element,Malondialdehyde and Immune Status in Drug Abuser Patients Undergoing Detoxification

Drug abuser patients (n = 104), age ranging from 19 to 42 years, were randomly recruited to investigate the serum levels of trace elements (Cu, Zn, Fe, and Mg), malondialdehyde (MDA), and immunoglobulin (IgG, IgA, and IgM) before and after clinical intervention. Control group also included 104 healthy individuals. Blood samples were analyzed for determining trace elements, MDA, and immunoglobulin using atomic absorption spectroscopy, Ultraviolet-Visible (UV-VIS) spectroscopy, and turbidimetry method, respectively. For serum level of Zn and Fe, the differences between the groups (before intervention, after intervention, and control) were not significant (p > 0.05). However, significant differences were found in serum copper levels between control group, drug abuser patients, and before and after intervention (p < 0.05). The concentration of Mg was found to be significantly higher (p = 0.007) in drug abuser patients than the controls, and after intervention, the level was restored to control value. A displacement of elemental homeostasis was observed in drug abuser patients compared to control, and it was improved after intervention. An increase in serum concentration of MDA was found in drug abuser patients compared to control subjects (p > 0.05) but was not statistically significant. After intervention, the concentration was restored to control value (p > 0.05). The serum concentrations of IgA and IgM were found to be significantly higher (p < 0.05) in drug abuser patients before intervention than the controls, and the level tended to be restored to control level after clinical intervention. Serum IgG level was found to be lower in drug abuser patients compared to controls and further declined significantly (p < 0.05) after intervention. These findings may suggest a possible imbalance in the levels of micronutrients, antioxidants, and immunoglobulin in drug abuser patients, which tend to be restored to control values after detoxification.     

Apyrase and 5′-nucleotidase Activities in Synaptosomes from the Cerebral Cortex of Rats Experimentally Demyelinated with Ethidium Bromide and Treated with Interferon-β

Apyrase and 5′-nucleotidase activities were analyzed in an ethidium bromide (EB) demyelinating model associated with interferon-β (IFN-β). The animals were divided in groups: I, control (saline); II, saline and IFN-β; III, EB and IV, EB and IFN-β. After 7, 15 and 30 days the animals (n=5) were sacrificed and the cerebral cortex was removed for synaptosome preparation and enzymatic assays. Apyrase activity using ATP as substrate increased in groups II, III and IV (P<0.001) after 7 days and in groups III and IV (P<0.001) after 15 days. Using ADP as substrate, an activation of this enzyme was observed in group III (P<0.05) after seven and 15 days. The 5′-nucleotidase activity increased in group III (P<0.05) after 7 days and in groups II, III and IV (P<0.001) after 15 days. After 30 days treatment, no significant alteration was observed in enzyme activities. Results showed that apyrase and 5′-nucleotidase activities are altered in demyelination events and that IFN-β was able to regulate the adenine nucleotide hydrolysis.     

Up-regulation of peroxisome proliferator-activated receptor gamma in radiation-induced heart injury in rats

To explore the expression level and the role of peroxisome proliferator-activated receptor gamma (PPAR-γ) in radiation-induced heart injury in a rat model, thirty-two Sprague–Dawley rats were divided into three groups (the control group, the 15-Gy irradiation group and the 18-Gy irradiation group). Experimental animals were exposed to radiation generated by a linear accelerator at the chest and killed after 3 months. Heart tissues from these animals were removed for Masson staining, PPAR-γ immunohistochemical staining, Western blot analysis and real-time polymerase chain reaction assay (RT-PCR). In addition, the protein expression of matrix metalloprotein-1 (MMP-1), tissue inhibitor of metalloproteinase-1 (TIMP-1) and transforming growth factor type beta1 (TGF-β1), all of which are associated with fibrosis, was measured. Masson staining revealed significant myocardial fibrosis, degeneration and necrosis in rats exposed to radiation. The results of immunohistochemical staining and Western blot analysis showed that PPAR-γ protein expression in hearts of the irradiation groups was significantly higher than in the control group, especially in myocardium and vascular endothelial (p < 0.05). RT-PCR results also showed a parallel increase in PPAR-γ mRNA expression in the heart of the irradiation groups compared with the control group (p < 0.05). The expression of MMP-1 protein was not significantly different in three groups (p > 0.05). The expression of TIMP-1 and TGF-β1 proteins was, however, higher in two irradiation groups than in the control group (p < 0.05). These data demonstrate that PPAR-γ expression is up-regulated on both mRNA and protein levels in heart injured by radiation. PPAR-γ may play an important role in radiation-induced heart injury.     

Metformin restores the correlation between serum-oxidized LDL and leptin levels in type 2 diabetic patients

Abstract

Introduction

Leptin has lipid peroxidation properties in healthy individuals. Here we aimed to study the correlation between serum-oxidized low-density lipoprotein (ox-LDL) and leptin levels in patients with type 2 diabetes. We also studied the effect of metformin therapy on the correlation between serum ox-LDL and leptin levels in patients with newly diagnosed diabetes.

Methods

We performed a cross-sectional study on two groups of patients with type 2 diabetes stratified according to (1) patients with newly diagnosed diabetes and (2) patients with long-standing diabetes plus healthy controls. Patients with newly diagnosed diabetes were followed for 3 months after the initiation of metformin therapy.

Results

Patients with type 2 diabetes had a higher serum ox-LDL, ox-LDL/LDL ratio, waist circumference, fasting blood sugars (FBSs), hemoglobin A1C (HbA1C), triglyceride, homeostatic model assessment of insulin resistance (HOMA-IR) and a lower serum leptin levels than controls. Serum ox-LDL, ox-LDL/LDL ratio (0.08 (0.08–0.12) vs. 0.06 (0.05–0.08), P < 0.001) and HOMA-IR (3.26 ± 0.23 vs. 2.93 ± 0.32; P < 0.01) were decreased when serum leptin levels (15.9 ± 1.6 vs. 21.4 ± 2.5, P < 0.01) were increased after 3 months of metformin therapy. This remained significant after multiple adjustments for age, body mass index, FBS, HbA1c, and HOMA-IR. Leptin was significantly correlated with ox-LDL/LDL ratio in controls (r = 0.78, P < 0.01), and in patients with newly diagnosed diabetes (r = 0.4, P < 0.05), after metformin therapy. There were not any correlation between leptin and ox-LDL/LDL ratio in patients with long-standing diabetes and patients with newly diagnosed diabetes before treatment.

Discussion

Metformin restores the positive correlation between serum ox-LDL and leptin levels in patients with type 2 diabetes.     

Inhibition of latent transforming growth factor-β1 activation by lentivirus-mediated short hairpin RNA targeting the CD36 gene in NR8383 cells

CD36, a cell surface receptor for thrombospondin-1 (TSP-1), is believed to interact with latent transforming growth factor-β1 (L-TGF-β1) thereby activating its fibrogenic bioactivity. In this study, a lentiviral vector expressing a short hairpin RNA (shRNA) targeting the rat CD36 gene (Lv-shCD36) is developed and tested. To observe the inhibitory effect of Lv-shCD36 on the activation of L-TGF-β1, a rat alveolar macrophage cell line (NR8383), infected with either Lv-shCD36 or Lv-shCD36-NC (non-silenced control lentivirus), was treated with 0.1 μg/ml bleomycin, which is known to stimulate alveolar macrophages to release increasing amounts of TGF-β1. The results show that Lv-shCD36 can suppress expression of CD36 mRNA and protein in bleomycin-treated NR8383 cells. By quantifying active and total TGF-β1 in the supernatant, it was discovered that the quantity of total TGF-β1 is not significantly different between the three groups, while the quantity and percent of active TGF-β1 in the Lv-shCD36 group was significantly lower than in either the bleomycin-treated group or the Lv-shCD36-NC group, respectively (P < 0.05). These results suggest that Lv-shCD36 can inhibit activation of L-TGF-β1 secreted in bleomycin-treated NR8383 cells by decreasing the expression of CD36 on the cell membrane, thereby reducing binding of CD36 to TSP-1.     

Serum and Testicular Trace Element Concentration in Rabbits at Different Ages

Trace elements such as Zinc (Zn), copper (Cu), iron (Fe), and selenium (Se) are essential for male fertility. The fertilizing capacity of most animals reduced with advancing age. The objective of the present work was to determine the serum and testicular levels of Zn, Cu, and Se in young (10 months old) and old (30 months old) rabbits. Blood and testicular samples were obtained from rabbits after their slaughter. All samples were digested by concentrated acids and analyzed for trace elements by flame emission atomic absorption spectrophotometer. The results showed that serum Zn and Cu were lower in old rabbits than young (P < 0.01). Serum Se was higher in old than young rabbits (P < 0.05). Testicular Cu and Se were significantly lower in old than young rabbits (P < 0.01). However, Zn level was higher in old compared to young rabbits (P < 0.01). The serum Zn and Se were higher than the testicular levels (P < 0.01). The testicular Cu in both ages was higher than the serum level (P < 0.01), which suggest a very important role for Cu in the process of spermatogenesis in rabbits.     

The Effect of Selenium Supplementation in the Prevention of DNA Damage in White Blood Cells of Hemodialyzed Patients: A Pilot Study

Patients with chronic kidney disease (CKD) have an increased incidence of cancer. It is well known that long periods of hemodialysis (HD) treatment are linked to DNA damage due to oxidative stress. In this study, we examined the effect of selenium (Se) supplementation to CKD patients on HD on the prevention of oxidative DNA damage in white blood cells. Blood samples were drawn from 42 CKD patients on HD (at the beginning of the study and after 1 and 3 months) and from 30 healthy controls. Twenty-two patients were supplemented with 200 μg Se (as Se-rich yeast) per day and 20 with placebo (baker's yeast) for 3 months. Se concentration in plasma and DNA damage in white blood cells expressed as the tail moment, including single-strand breaks (SSB) and oxidative bases lesion in DNA, using formamidopyrimidine glycosylase (FPG), were measured. Se concentration in patients was significantly lower than in healthy subjects (P < 0.0001) and increased significantly after 3 months of Se supplementation (P < 0.0001). Tail moment (SSB) in patients before the study was three times higher than in healthy subjects (P < 0.01). After 3 months of Se supplementation, it decreased significantly (P < 0.01) and was about 16% lower than in healthy subjects. The oxidative bases lesion in DNA (tail moment, FPG) of HD patients at the beginning of the study was significantly higher (P < 0.01) compared with controls, and 3 months after Se supplementation it was 2.6 times lower than in controls (P < 0.01). No changes in tail moment was observed in the placebo group. In conclusion, our study shows that in CKD patients on HD, DNA damage in white blood cells is higher than in healthy controls, and Se supplementation prevents the damage of DNA.     

Enhanced β-adrenergic receptors in the brain and pancreas during pancreatic regeneration in weanling rats

Adrenergic stimulation has an important role in the pancreatic β-cell proliferation and insulin secretion. In the present study, we have investigated how sympathetic system regulates the pancreatic regeneration by analyzing Epinephrine (EPI), Norepinephrine (NE) and β-adrenergic receptor changes in the brain as well as in the pancreas. EPI and NE showed a significant decrease in the brain regions, pancreas and plasma at 72 hrs after partial pancreatectomy. We observed an increase in the circulating insulin levels at 72 hrs. Scatchard analysis using [³H] propranolol showed a significant increase in the number of both the low affinity and high affinity β-adrenergic receptors in cerebral cortex and hypothalamus of partially pancreatectomised rats during peak DNA synthesis. The affinity of the receptors decreased significantly in the low and high affinity receptors of cerebral cortex and the high affinity hypothalamic receptors. In the brain stem, low affinity receptors were increased significantly during regeneration whereas there was no change in the high affinity receptors. The pancreatic β-adrenergic receptors were also up regulated at 72 hrs after partial pancreatectomy. In vitro studies showed that β-adrenergic receptors are positive regulators of islet cell proliferation and insulin secretion. Thus our results suggest that the β-adrenergic receptors are functionally enhanced during pancreatic regeneration, which in turn increases pancreatic β-cell proliferation and insulin secretion in weanling rats.     

Design of a controlled release system of OP-1 and TGF-β1 based in microparticles of sodium alginate and release characterization by HPLC-UV

A new system for sustained release of growth factors, such as osteogenic protein 1 (OP-1) and transforming growth factor β1 (TGF-β1), intended to repair and promote dental tissue regeneration in rats was designed and characterized in this work. The release system was made with microparticles of sodium alginate, produced by ionic gelling dripping technique. The release profiles of OP-1 and TGF-β1 from biopolymer matrix were determined by high-performance liquid chromatography (HPLC), and with this purpose, an HPLC-UV method was developed. About 80% of each growth factor was released in the first 24 h, reaching almost 100% in 168 h. The system was tested during the tissue repair in rat molars in comparison with calcium hydroxide and both growth factors not encapsulated. The dentin sialoprotein (DSP) was used as a repair marker. It was detected by immunohistochemistry, after 14- and 28-d post-treatment. X ² test (p ≤ 0.001) and Fisher exact test (p ≤ 0.05) were applied for assessment of the amount of immunostaining. The treatment with encapsulated OP-1 showed an increased DSP immunostaining after 14 d and did not find any significant difference with the immunostaining observed for calcium hydroxide treatment. Treatment with TGF-β1 did not show significant difference with calcium hydroxide. Treatment with both factors OP-1 and TGF-β1 showed higher DSP immunostaining in comparison with calcium hydroxide treatment. In conclusion, the combination of both growth factors encapsulated showed more DSP immunostaining in comparison with each one separated, either encapsulated or not.