Effects of Lucilia sericata on wound healing in streptozotocin-induced diabetic rats and analysis of its secretome at the proteome level.

The use of Lucilia sericata larvae on the healing of wounds in diabetics has been reported. However, the role of the excretion/secretion (ES) products of the larvae in treatment of diabetic wounds remains unknown. This study investigated whether application of the ES products of L. sericata on the wound surface could improve the impaired wound healing in streptozotocin-induced diabetic rats. Additional analysis was performed to understand proteome content of L. sericata secretome to understand ES contribution at the molecular level. For this purpose, full-thickness skin wounds were created on the backs of diabetic and control rats. A study was conducted to assess the levels of the ES-induced collagen I/III expression and to assay nuclear factor κB (NF-κB) (p65) activity in wound biopsies and ES-treated wounds of diabetic rat skin in comparison to the controls. The expression levels of collagen I/III and NF-κB (p65) activity were determined at days 3, 7, and 14 after wounding using immunohistological analyses and enzyme-linked immunosorbent assay technique. The results indicated that treatment with the ES extract increased collagen I expressions of the wound control and diabetic tissue. But the increase in collagen I expression in the controls was higher than the one in the diabetics. NF-κB (p65) activity was also increased in diabetic wounds compared to the controls, whereas it was decreased in third and seventh days upon ES treatment. The results indicated that ES products of L. sericata may enhance the process of wound healing by influencing phases such as inflammation, NF-κB (p65) activity, collagen synthesis, and wound contraction. These findings may provide new insights into understanding of therapeutic potential of ES in wound healing in diabetics.

The Correlation of Increased CRP Levels with NFKB1 and TLR2 Polymorphisms in the Case of Morbid Obesity.

Morbid obesity (MO) is associated with an increase in circulating levels of systemic acute phase proteins such as C-reactive protein (CRP). Toll-like receptor is possible candidate for inflammatory responses which is mainly mediated by NFKB1. The aim of this study was to investigate the relationship between NFKB1 and Toll-like receptor (TLR) 2 polymorphisms and the risk of MO in a Turkish population in the context of CRP serum levels which may contribute to susceptibility to the disease. We analysed the distribution of NFKB1-94 ins/del ATTG rs28362491 and TLR2 Arg753Gln rs5743708 polymorphisms using PCR-RFLP method and CRP serum levels using ELISA method in 213 MO and 200 healthy controls. The frequency of the ins/ins genotype and ins allele of rs28362491 was significantly higher in the patients compared to control group (P: 0.0309; P: 0.0421, respectively). Additionally, the frequency of GG genotype and G allele of rs5743708 was found to be statistically higher in the patient group (P: 0.0421; P < 0.0001, respectively). In addition, serum CRP levels (>20 mg/l) in MO patients with ins/ins genotype were significantly higher than in patients with del/ins genotype (P: 0.0309). Serum CRP levels were also higher in MO patients with GG genotype and G allele (P: 0.0001). According to combined analysis, the wild type of rs28362491 and rs5743708 polymorphisms (ins/ins/GG genotype) was also significantly higher in the patient group versus the control group when compared with the combined ins/ins/GA and del/ins/GA genotype (P < 0.0001). Therefore, our findings suggest that rs28362491 and rs5743708 polymorphisms were significantly associated with MO disease through acting by modulating serum CRP levels.

Association of NFKB1 and NFKBIA polymorphisms in relation to susceptibility of Behçet's disease.

Behçet's disease (BD) is a chronic inflammatory autoimmune disease. Although raised levels of proinflammatory cytokines in BD have been reported, the pathogenesis is still unknown. The aim of this study was to investigate the association of NFKB1 and NFKBIA polymorphisms and their single and combined analysis effects on susceptibility of BD in Turkish population. We analysed the distribution of NFKB1 -94 ins/del ATTG (rs28362491) and NFKBIA 3' UTR A→G (rs696) polymorphisms using PCR-RFLP method in 89 patients with BD and 190 controls in this population. Statistical analysis of the results was performed by calculating OR, and 95% CI via χ(2) test and using Bonferroni correction. According to the significant results of both single and combined genotype analysis, the frequencies of ins/ins genotype and ins allele of rs28362491 were significantly higher in patients with BD (Pc = 0.003, 0.004, respectively). Also, higher frequencies of the rs696 variant containing AA genotype was found in patients with BD (Pc = 0.0033), whereas no statistical significant differences in distribution of the alleles of rs696 polymorphism in patients and controls. In addition, according to the combined genotype analysis, the wild type of both rs28362491 and rs696 polymorphisms (ins/ins/AA genotype) was also significantly higher in BD cases (Pc = 0.044). Our findings prove that both single and combined genotype analysis of rs28362491 and rs696 polymorphisms indicate that the wild genotypes of both two SNPs (ins/ins and AA genotypes) and ins/ins/AA combined genotype are strongly associated with enhanced risk of BD in a Turkish population.

Age-related changes on glucose transport and utilization of human erythrocytes: effect of oxidative stress.

BACKGROUND: Normal aging is associated with an impairment in glucose homeostasis. METHODS: In order to investigate the effect of aging on glucose transport and utilization in erythrocytes, the transport and utilization of glucose were measured in erythrocytes from 10 young (mean age 26 +/- 3 years) and 10 elderly (mean age 70 +/- 7 years) healthy individuals. In addition, the glucose transport and utilization were also measured in the presence of cumene hydroperoxide (CumOOH), a toxic organic hydroperoxide that is known to induce oxidative stress. RESULTS: The levels of glucose transport and utilization were significantly lower in the elderly group than the young group (p < 0.05). The glucose transport and utilization were not altered by CumOOH treatment in either young or elderly individuals. CONCLUSION: These results indicate an age-related decrease in the both glucose transport and utilization in erythrocytes.

The modulation of glucocorticoid receptor content by 3-O-methyl-D-glucose transport in human mononuclear leukocyte in obesity.

Glucocorticoid receptors (GR) and 3-O-methyl-D glucose (3-O-MG) transport were determined in mononuclear leukocytes (MNL) from 11 abdominal obese subjects, 10 pituitary-dependent Cushing's syndrome (Cushing's disease) and 10 healthy controls. Using a whole-cell competitive binding assay and 3H-dexamethasone as tracer, MNL of abdominal obese subjects were found to have 4855 +/- 1389 sites/cell which was significantly lower (p < 0.05) than controls (6234 +/- 1568 sites/cell), although no significant difference was found in the mean serum cortisol level. Their mean Kd (affinity) was also significantly lower than that found in the healthy controls (obese Kd:2.92 +/- 0.84 nmol/l, control Kd: 4.55 +/- 0.67 nM, p < 0.05). On the other hand, the receptor characteristics in Cushing's disease patients were within the normal range. At the same time, 3-O-MG transport was determined in the same subjects. In Cushing's disease, 3-O-MG transport was within the normal range, whereas in abdominal obesity this value was significantly lower than the healthy controls (abdominal obese: 31.90 +/- 8.20; control: 46.26 +/- 12.91 fmol/10(6) cell, min, p < 0.05). We also found a positive correlation between 3-O-MG transport and GR binding capacity in abdominal subjects (r = 0.89, p < 0.001), however we did not find such a correlation in Cushing's disease (r = 0.60, p > 0.05). These results indicated that, in abdominal obesity, the GR binding capacity in MNL is influenced by the changes in glucose transport.

The effect of metformin on insulin receptors and lipid peroxidation in alloxan and streptozotocin induced diabetes.

Biguanides are used for the treatment of non-insulin dependent diabetes mellitus but there is no evidence for an improving action of biguanide on the enhancement of peripheral glucose disposal in type 1 diabetes. It is known that biguanide agents reduce the oxidation of free fatty acids. Using alloxan and streptozotocin (STZ) induced diabetic rats as a model for type 1 diabetes mellitus, we measured insulin binding capacity and plasma lipid peroxidation levels before and after metformin induction. There was a significant increase in insulin binding capacity and lipid peroxidation levels in alloxan and STZ diabetes compared to controls. We examined the effect of metformin on alloxan and STZ-induced diabetic rats. In alloxan-induced diabetes metformin (Met) treatment led to an increase in insulin receptor number in liver plasma membranes (before Met: 46.50 +/- 2.69, after Met: 76.00 +/- 3.39 fmol/mg, p < 0.001) and a decrease in plasma lipid peroxidation levels compared to the non-treated group (before Met: 1.85 +/- 0.53, after Met: 1.10 +/- 0.09 nmol MDA/ml, p < 0.05). In STZ-induced diabetic rats metformin treatment did not change the lipid peroxidation levels (before Met: 1.26 +/- 0.31, after Met: 1.38 +/- 0.44 nmol MDA/ml, p > 0.05) whereas it did increase the receptor numbers (before Met: 41.60 +/- 4.33, after Met: 63.40 +/- 8.64 fmol/mg, p < 0.002).

The in vitro effects of captopril on the levels of lipid peroxidation and glutathione of erythrocytes in type II diabetes.

Increase in lipid peroxidation (LP) is an indirect marker of free radical activation. The products of LP (malonyldialdehyde: MDA) are increased in diabetic patients, particularly those with angiopathy. Free radicals are eliminated by cellular enzymes such as superoxide dismutase, catalase and glutathione peroxidase. In this study, the effect and the mechanism of action of captopril, and angiotensin converting enzyme (ACE) inhibitor, on lipid peroxidation in erythrocytes from diabetics was investigated. LP and glutathione were studied in 10 type II diabetics (mean age: 57 +/- 10 yr, duration of diabetes: 12 +/- 6 yr) and in 10 healthy subjects (mean age: 30 +/- 5 yr). Lipid peroxidation levels were 20.69 +/- 4.68 MDA% in diabetics and 9.62 +/- 1.87 MDA% in normal subjects. The LP in erythrocytes of type II diabetics was decreased by the increasing concentrations of captopril (before captopril: 20.69 +/- 4.68, after captopril: (2 x 10(-5) M) 16.68 +/- 7.49 MDA%; (4 x 10(-5) M) 14.17 +/- 7.65 MDA%; (6 x 10(-5) M) 12.33 +/- 2.8 MDA%). No difference was found in the inhibition of LP between the captopril concentrations of 6 x 10(-5) M and 10 x 10(-5) M. After preincubation with captopril, the glutathione level did not change significantly in the diabetic and normal erythrocytes. Preincubation with 2-6 x 10(-5) M captopril showed no effect in the normal group (p > 0.05) but 10 x 10(-5) M captopril reduced lipid peroxidation (p < 0.01). In our study, the high levels of lipid peroxidation in erythrocytes from diabetic patients were decreased after preincubation with captopril. Decrease in the level of lipid peroxidation in vitro was independent of the glutathione level. Crosslink binding between MDA and captopril is suggested.