Topical metformin accelerates wound healing by promoting collagen synthesis and inhibiting apoptosis in a diabetic wound model.

The wound healing process, which is a pathophysiological process that includes various phases, is interrupted in diabetes due to hyperglycemia, and since deterioration occurs in these phases, a normal healing process is not observed. The aim of the current study is to investigate the proliferative and antiapoptotic effects of metformin on wound healing after topical application on diabetic and non-diabetic wounds. For this purpose, we applied metformin topically on the full-thickness excisional wound model we created in diabetic and nondiabetic groups. We investigated the effects of metformin on the apoptotic index by the Terminal deoxynucleotidyl transferase mediated dUTP Nick-End Labeling method and on collagen-I, collagen-III, p53, and c-jun expression levels by quantitative reverse transcription polymerase chain reaction technique in wound biopsy tissues. Our results showed that c-jun and p53 mRNA levels and apoptotic index increased with the effect of diabetes, while collagen synthesis was disrupted. As a result of the study, we showed that metformin increases cellular proliferation and has anti-apoptotic effects by increasing collagen-I/III expression and decreasing p53/c-jun level, especially in diabetic wounds and also in normal wounds. In conclusion, the topical effect of metformin on diabetic wounds reversed the adverse effects caused by diabetes, increasing the wound healing rate and improving the wound repair process.

Fas and microRNAs Variations as a Possible Risk for Behçet Disease.

BACKGROUND: Behçet disease (BD) belongs to a disease family that has a transparent borderline between autoinflammatory and autoimmune disorders. Fas and some miRNAs have revealed to display remarkable roles in both autoimmune and autoinflammatory processes, and they can play important roles in defective apoptosis in BD. We investigated the association of the susceptibility of BD with Fas, miRNA variations, and their both single and combined presence in a Turkish population as a case-control study. METHODS: The distributions of FAS-670 A>G rs1800682, mir146a rs2910164, and mir196a rs11614913 polymorphisms are analyzed with the polymerase chain reaction-restriction fragment length polymorphism method in 115 BD patients and 220 controls in 6-month period. RESULTS: Statistical analysis indicates that in the case of Fas-670 A/G rs1800682, AA genotype and A allele have a protective role in BD (p = 0.0004 and p = 0.0009, respectively). The dominant model (AA + AG/GG) also displays a protective effect on BD unlike the recessive model (p = 0.03). In addition, both homozygous genotype (CC) of rs2910164 of mir-146a (p = 0.04) and the dominant model (CC + CG vs. GG) have protective effects on BD unlike the recessive model (p < 0.0001). Both mir-196a2 rs1800682 polymorphism and combined genotype analysis of rs1800682-rs2910164 and rs1800682-rs11614913 gave no statistically significant differences within the groups for genotypes and either of the alleles (p > 0.05). CONCLUSIONS: These findings indicate that both Fas rs1800682 and mir-146a rs2910164 variants might be important factors participating in the protection against BD in the Turkish population.

NF-κB as the mediator of metformin's effect on ageing and ageing-related diseases.

Ageing can be defined as the progressive failure of repair and maintenance systems with a consequent accumulation of cellular damage in nucleic acids, proteins, and lipids. These various types of damage promote ageing by driving cellular senescence and apoptosis. The nuclear factor-kappa B (NF-kB) pathway is one of the key mediators of ageing and this pathway is activated by genotoxic, oxidative and inflammatory stress, and regulates expression of cytokines, growth factors, and genes that regulate apoptosis, cell-cycle progression, and inflammation. Therefore, NF-kB is increased in a variety of tissues with ageing, thus the inhibition of NF-kB leads to delayed onset of ageing-related symptoms and pathologies such as diabetes, atherosclerosis, and cancer. Metformin is often used as an anti-diabetic medication in type 2 diabetes throughout the world and appears to be a potential anti-ageing agent. Owing to its antioxidant, anticancer, cardio-protective and anti-inflammatory properties, metformin has become a potential candidate drug, improving in the context of ageing and ageing-related diseases. An inappropriate NF-kB activation is associated with diseases and pathologic conditions which can impair the activity of genes involved in cell senescence, apoptosis, immunity, and inflammation. Metformin, inhibiting the expression of NF-kB gene, eliminates the susceptibility to common diseases. This review underlines the pleiotropic effects of metformin in ageing and different ageing-related diseases and attributes its effects to the modulation of NF-kB.

Variations in Toll-like receptor and nuclear factor-kappa B genes and the risk of glioma.

PURPOSE: Glioblastoma (GBM) is the most aggressive primary brain tumour in the adult nervous system and is associated with a poor prognosis. NF-KB activation is an important driver of the malignant phenotype that confers a negative prognosis in patients with GBM. NF-KB plays a role in Toll-like Receptors (TLR)-induced tumourigenesis. The aim of the present study was to investigate the association of a promoter region polymorphism of NFKB1 gene encoding the p50 subunit of NF-KB, namely -94ins/del ATTG, the most widely discussed the TLR2 Arg753Gln, TLR4Asp299Gly and TLR4Thr399Ile polymorphisms, their combined effects, and the glioma risk. METHODS: A group of 120 Glioma patients and 225 control subjects were screened for these four polymorphisms using the PCR-RFLP method. RESULTS: Statistical analysis indicates that the ins/ins genotype of NFKB -94ins/delATTG (p=0.003), and the AA genotype of TLR4Asp299Gly (p < 0.001) are risk factors for glioma and people carrying the ins allele have an approximately 1.47 times susceptibility risk of glioma whereas GG genotype of TLR2Arg753Gln seems to be protective against glioma (p = 0.002). Combined genotype analysis showed that del/ins-GG genotype of TLR2Arg753Gln-NFKB1, del/ins + GG genotype of TLR4Asp299Gly-NFKB1, del/ins-CC genotype of TLR4Thr399Ile-NFKB1 were risk factors for glioma development. CONCLUSION: NFKB1 -94ins/delATTG and TLR4Asp299Gly polymorphisms are associated with increased glioma cancer risk in a Turkish population.

Association of IL-23R and IL-10 variations with Behçet disease: a genetic analysis study.

Behçet disease (BD) is an autoimmune and autoinflammatory disease mainly affecting the Silk Road countries. The interindividual severity of BD depends on differences in the polymorphic profiles of the patients. One of the most prominent markers, HLA-B51 positivity, is also observed in 40-60% of patients with BD on the Silk Road. Inflammatory markers such as interleukin 10 (IL-10) and interleukin 23 receptor (IL-23R) are also widely associated with BD etiology. The polymorphisms on these genes may change the susceptibility to BD. In this case-control study, we assessed the associations of IL-10 rs3024498 and IL-23R rs10889677 single-nucleotide polymorphisms (SNPs) with BD susceptibility, if any. Two hundred eighty HLA-B51-positive patients with BD and 300 healthy controls were genotyped for these SNPs using RFLP-PCR. The chi-square test was used for genotyping. We found that IL-23R rs10889677 CC and IL-10 rs3024498 CT genotype frequencies were higher in the BD group than in the control group (p < 0.0001 and p = 0.0293, respectively). The recessive model (AA + CC vs. AC) and combined genotype (AC + CT) results were also statistically significant (p < 0.0001 and p = 0.0364, respectively). We conclude that IL-23R rs10889677 and IL-10 rs3024498 SNPs may be associated with the susceptibility to BD.

Metformin reverses the effects of high glucose on human dermal fibroblasts of aged skin via downregulating RELA/p65 expression.

Metformin has been successfully used as an anti-aging agent but exact molecular mechanisms of metformin in anti-aging remain unknown. Hyperglycemia during skin aging not only causes oxidative damage to cellular macromolecules, like dermal collagen, but also modulates the activation of transcription factor nuclear factor kappa B (NF-kB). We aimed to investigate in vitro effects of high glucose (HG) and metformin treatment on proliferation and apoptosis of human primary dermal fibroblasts (HDFs), and the expression of COL1A1, COL3A1, and RELA/p65 genes. Effects of normal glucose (5.5 mM) and HG concentration (50 mM HG) on HDFs, with two doses of metformin (50 µM and 500 µM), were investigated by immunostaining. Apoptotic levels were analyzed by flow cytometry. Expression of COL1A1, COL3A1, and RELA/p65 genes was measured by quantitative real-time PCR. The proliferation of HDFs was decreased significantly (P < 0.01) and expression of COL1A1 was downregulated by HG without metformin, whereas proliferation was elevated and expression was upregulated with 500 µM metformin + HG compared to 5.5 mM glucose (P < 0.05). The expression of COL3A1 and RELA/p65 were upregulated (P < 0.01 for COL3A1), and percentage of late apoptotic cells increased significantly by HG without metformin (P < 0.001) while it decreased in two concentrations of metformin dramatically compared with 5.5 mM glucose (P < 0.01 for expressions and < 0.001 for apoptosis). Metformin not only significantly downregulated RELA/p65 expression, but also inhibited the apoptosis of HDFs from aged human skin at toxic glucose concentrations which could be inversely mediated via COL1A1 and COL3A1 expression.

Anti-cancer effect of metformin on the metastasis and invasion of primary breast cancer cells through mediating NF-kB activity.

Current evidence strongly suggests that aberrant activation of the nuclear factor kappa B (NF-kB) signaling cascade is connected to carcinogenesis. The matrix metalloproteinases (MMP) which are also the key agents for tumor metastasis may be potent candidates for tumor diagnosis in clinics. In this in vitro study, we hypothesized that metformin with an effective dose can inhibit tumor cell proliferation and metastasis by modulating the expressions of MMP-2 and -9 and interfering with NF-kB signaling in primary breast cancer cells (PBCCs). 300 000 cells per ml were obtained from biopsies of breast tumors from five human donors. The cell viability and proliferation were tested. Immunocytochemistry was performed for MMP-2, MMP-9, and NF-kB, and enzyme-linked immunosorbent assay for NF-kB activity, quantitative real-time PCR for RELA/p65, IkBα, MMP-2, and MMP-9. Three different doses of metformin (5, 10, and 25 mM) (Met) reduced the viability and proliferation of PBCCs in a dose-dependent manner, maximum inhibition was observed at 25 mM Met. The expression of RELA/p65 was not affected by 25 mM Met. Nuclear immunoreactivity and activity of NF-kB reduced while cytoplasmic NF-kB (p65) elevated by 25 mM Met compared to non-treatment (P <  0.05). The expression and immunoreactivity of MMP-9 but not MMP-2 were decreased by 25 mM Met treatment, compared with the non-treatment (P <  0.05). Metformin may have an essential antitumor role in the invasion and metastasis pathways of PBCCs by downregulating the MMP-9 expression blocking both the activity and nuclear translocation of NF-kB.

Regulation of MMP 2 and MMP 9 expressions modulated by AP-1 (c-jun) in wound healing: improving role of Lucilia sericata in diabetic rats.

AIMS: Lucilia sericata larvae have been successfully used on healing of wounds in the diabetics. However, the involvement of the extraction/secretion (ES) products of larvae in the treatment of diabetic wounds is still unknown. Activator protein-1 (AP-1) transcription, composed of c-jun and c-Fos proteins, has been shown to be the principal regulator of multiple MMP transcriptions under a variety of conditions, also in diabetic wounds. Specifically, MMP-2 and MMP-9's transcriptions are known to be modulated by AP-1. c-jun has been demonstrated to be a repressor of p53 in immortalized fibroblasts. The aim of the present study is to investigate the effects of L. sericata ES on the expression of AP-1 (c-jun), p53, MMP-2, and MMP-9 in wound biopsies dissected from streptozotocin induced diabetic rats. METHODS: The expression levels of MMP-2, MMP-9, c-jun and p53 in dermal tissues were determined at days 0, 3, 7 and 14 after wounding, using immunohistochemical analysis and quantitative real-time PCR. RESULTS: The treatment with ES significantly decreased through inflammation-based induction of MMP-2 and MMP-9 expression levels in the wounds of diabetic groups, compared to control groups at the third day of wound healing. At the 14th day, there were dramatic decreases in expression of c-jun, MMP-9, and p53 in ES-treated groups, compared to the diabetic group (P < 0.001, P < 0.05 and P < 0.01, respectively). CONCLUSION: ES products of L. sericata may enhance the process of wound healing in phases of inflammation, proliferation, and re-epithelization, essentially via regulating c-jun expression and modulating MMP-2 and MMP-9 expressions.

Genetic Variation in NFKB1 Gene Influences Liver Enzyme Levels in Morbidly Obese Women.

BACKGROUND: Morbid obesity (MO), characterized by low-grade inflammation, is associated with increased C-reactive protein (CRP). NF-KB is a candidate factor for inflammatory responses in inflammatory diseases such as obesity. The objective of our study was to investigate the relationship between NFKB1 gene variations and the risk of MO in the context of the high/normal level of liver enzymes such as Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT), and alkaline phosphatase (ALP). METHODS: We analyzed the distribution of NFKB1 -94 ins/del ATTG (rs28362491) polymorphism using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)and liver enzymes serum levels using ELISA in 182 MO patients with CRP level ≥20 mg/L and 200 healthy controls in a female Turkish population. RESULTS: We found that having ins/ins genotype of rs28362491 is a risk factor in both high level and normal level liver enzymes of ALT (P = 0.0335, P = 0.0134), AST (P = 0.0285, P = 0.0113) and ALP (P = 0.0079, P = 0.0363) whereas having ins/ins genotype of rs28362491 is a risk factor in only high-level liver enzyme of GGT (P = 0.0003). CONCLUSION: Our results suggest that ins/ins genotype of SNP rs28362491 is linked to MO with high-level ALT, AST, ALP, and GGT.

The protective effects of metformin in an in vitro model of aging 3T3 fibroblast under the high glucose conditions.

Metformin is the most widely used anti-diabetic drug in the world. It reduces advanced glycation end product (AGEs)-induced ROS generation in high glucose condition. Protein glycation contributes to skin aging as it deteriorates the existing collagen by crosslinking. The progressive increase of AGE during aging not only causes oxidative damage to cellular macromolecules but also modulates the activation of transcription factors nuclear factor kappa-B(NF-kB). However, it is still unclear whether metformin can change collagen production and NF-kB activity induced by high glucose conditions in 3T3 fibroblast. The effects of metformin on proliferation, apoptosis, and collagen levels and NF-kB activity of in vitro cell aging model of 3T3 fibroblast cells in high glucose conditions. At first, we investigated the effects of 50 mM high glucose concentration, with or without metformin, on 3T3 fibroblast proliferation, by BrdU immunostaining for cell proliferation. Apoptotic levels were analyzed by flow cytometric assay. NF-kB(p65) activity was measured by transcription factor assay kit and collagen I and III levels by Collagen Estimation Assay through ELISA. We observed that metformin exposure leads to decreased apoptosis levels and increased proliferation of 3T3 fibroblast in high glucose media. We also determined that metformin exposure leads to increased production of collagen I-III and decreased activation of NF-kB(p65) activity. The data are consistent with the observation that metformin has a protective effect in this in vitro model of aging 3T3 fibroblasts under high glucose conditions inducing cell proliferation, collagen I and III production, protection from apoptosis, and reducing NF-kB(p65) activity.

NFKB1 rs28362491 and pre-miRNA-146a rs2910164 SNPs on E-Cadherin expression in case of idiopathic oligospermia: A case-control study.

BACKGROUND: A notable proportion of idiopathic male infertility cases is accompanied by oligozoospermia; and yet, the molecular mechanisms of fertilization problem underlying this defect are still unclear. Epithelial cadherin has been involved in several calcium-dependent cell-to-cell adhesion events; however, its participation in gamete interaction has also not been fully investigated. OBJECTIVE: The aim was to investigate the changes in the expression of E-cadherin, based on the frequency of Single nucleotide polymorphisms in Nuclear Factor Kappa-B 1 and pre-mir-146a in oligospermic men. MATERIALS AND METHODS: In this case-control study, semen and blood samples of 131 oligospermic men as the case group and 239 fertile healthy men as the control group were analyzed. Variants single nucleotide polymorphisms rs28362491 and rs2910164 were performed using polymerase chain reaction-restriction fragment length polymorphism method and E-cadherin expression were determined by immunoprecipitation studies. RESULTS: ins/ins genotype of rs28362491 was determined as a risk factor for idiopathic oligospermia by 1.73 times (p=0.0218), whereas no significant differences were found between the groups concerning pre-mir-146a rs2910164 polymorphism (p=0.2274 in case of GC genotype and p=0.9052 in case of GG genotype). Combined genotype analysis results did not show any notable differences between the multiple comparisons of 28362491-rs2910164 in oligospermic men and control groups. In addition, E-cadherin expression of oligospermic men with ins/ins genotype was significantly lower than patients with del/ins genotype (p=0.0221). E-cadherin expression level was low in oligospermic men with respect to the control group in presence of ins/ins genotype of NFKB1 gene. CONCLUSION: These results suggest that ins allele prevents binding of surface proteins to spermatozoa, leading to a low affinity of sperm-oocyte interaction in oligospermic men.