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.

Topical application of metformin accelerates cutaneous wound healing in streptozotocin-induced diabetic rats.

BACKGROUND: Diabetic chronic wound, which is one of the diabetic complications caused by hyperglycemia, characterized by prolonged inflammation has become one of the most serious challenges in the clinic. Hyperglycemia during diabetes not only causes prolonged inflammation and delayed wound healing but also modulates the activation of nuclear factor-kappa B (NF-κB) and the expression of matrix metalloproteinases (MMPs). Although metformin is the oldest oral antihyperglycemic drug commonly used for treating type 2 diabetes, few studies have explored the molecular mechanism of its topical effect on wound healing. Therefore, we aimed to investigate the molecular effects of topical metformin application on delayed wound healing, which's common in diabetes. METHODS AND RESULTS: In this context, we created a full-thickness excisional wound model in Wistar albino rats and, investigated NF-κB p65 DNA-binding activity and expression levels of RELA (p65), MMP2 and MMP9 in wound samples taken on days 0, 3, 7, and 14 from diabetic/non-diabetic rats treated with metformin and saline. As a result of our study, we showed that topically applied metformin accelerates wound healing by suppressing NF-κB p65 activity and diminishing the expression of MMP2 and MMP9. CONCLUSIONS: Diabetic wounds treated with metformin healed even faster than those in the control group that mimicked standard wound healing.

A molecular approach to maggot debridement therapy with Lucilia sericata and its excretions/secretions in wound healing.

Chronic wounds caused by underlying physiological causes such as diabetic wounds, pressure ulcers, venous leg ulcers and infected wounds affect a significant portion of the population. In order to treat chronic wounds, a strong debridement, removal of necrotic tissue, elimination of infection and stimulation of granulation tissue are required. Maggot debridement therapy (MDT), which is an alternative treatment method based on history, has been used quite widely. MDT is an efficient, simple, cost-effective and reliable biosurgery method using mostly larvae of Lucilia sericata fly species. Larvae can both physically remove necrotic tissue from the wound site and stimulate wound healing by activating molecular processes in the wound area through the enzymes they secrete. The larvae can stimulate wound healing by activating molecular processes in the wound area through enzymes in their excretions/secretions (ES). Studies have shown that ES has antibacterial, antifungal, anti-inflammatory, angiogenic, proliferative, hemostatic and tissue-regenerating effects both in vivo and in vitro. It is suggested that these effects stimulate wound healing and accelerate wound healing by initiating a direct signal cascade with cells in the wound area. However, the enzymes and peptides in ES are mostly still undefined. Examining the molecular content of ES and the biological effects of these ingredients is quite important to illuminate the molecular mechanism underlying MDT. More importantly, ES has the potential to have positive effects on wound healing and to be used more as a therapeutic agent in the future, so it can be applied as an alternative to MDT in wound healing.

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.

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.

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.

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.

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.

A Role for Heterozygosity of NF-𝜠B1 rs28362491 Polymorphism in Patients with Idiopathic Oligospermia.

BACKGROUND: Nuclear factor-kappa B (NF-𝜅B) activation and its inhibition by NF-𝜅B inhibitor (I𝜅B) have been functionally linked to germ cell apoptosis, which may affect human infertility. We hypothesized a possible relationship between the NF-𝜅B1-94ins/del ATTG (rs28362491) and NF-𝜅BIA 3'UTR A→G (rs696) polymorphism, which are common polymorphisms and the susceptibility to oligospermia in the context of the sperm apoptosis. METHODS: In order to evaluate this association, we studied the polymorphisms and sperm apoptosis rates of 114 men with idiopathic oligospermia, as well as 130 normospermic men, using PCR-RFLP and TUNEL staining methods, respectively. RESULTS: Univariate analysis revealed that heterozygous ID genotype at the NF-𝜅B1 -94ins/del ATTG polymorphism is associated with an approximately 2.4-fold reduced risk of oligospermia (P = 0.006, 95% confidence intervall = 1.34 - 4.13). However, the genotype and allele frequencies of NF-𝜅BIA 3'UTR A→G polymorphism, and the genotype frequencies of all possible rs28362491/rs696 genotype combinations did not show any significant differences between oligospermic and normospermic men. Furthermore, neither polymorphism appeared to affect sperm apoptosis, although the sperm apoptosis index was detected to be significantly higher in the oligospermic patients compared with those in the controls (P < 0.05). CONCLUSION: Our findings suggested that the heterozygosity of rs28362491 in the NF-𝜅B1 gene may have a protective effect against oligospermia and could modify the susceptibility of oligospermia in a group with idiopathic male infertility in a Turkish population.

Investigation of poly (ADP-ribose) polymerase-1 genetic variants as a possible risk for allergic rhinitis.

Recent studies point toward the involvement of poly (ADP-ribose) polymerase-1 (PARP-1) in the pathogenesis of allergic airway inflammation, such as asthma and allergic rhinitis (AR). It has been suggested that inhibition of PARP-1 provides significant protection against systemic or tissue inflammation in animal models. The objective of this study was to investigate whether single-nucleotide polymorphisms of PARP-1 gene are associated with genetic susceptibility to AR. We studied the effect of promoter variations and Val762Ala polymorphism of the PARP-1 gene on the risk for developing AR in a case-control association study with 110 RA patients and 130 control subjects in a Turkish population. The polymorphisms of 410 C/T, -1672G/A, and Val762Ala in the PARP-1 gene were analyzed using the polymerase chain reaction-restriction fragment length polymorphism method. Haplotype analysis of these groups was also performed. The results were statistically analyzed by calculating the odds ratio (OR) and their 95% confidence intervals using χ(2) tests. The heterozygote genotype of the promoter polymorphism (-1672) was significantly found to be associated with susceptibility to AR (OR: 0.56) among the tested single-nucleotide polymorphisms. Haplotypes of PARP-1 -410, -1672, and 762 were not associated with an increased risk for AR. These results raise the possibility that the promoter (-1672) polymorphism of the PARP-1 gene may be a risk factor for AR.

Glioma risk associates with polymorphisms of DNA repair genes, XRCC1 and PARP1.

Cancer develops through interactions between polygenic and environmental factors, and changes in DNA repair pathway can increase susceptibility to tumours. XRCC1 and PARP1 are two proteins that act cooperatively in base excision repair (BER) of DNA. The polymorphisms of genes coding these proteins may effect their action in BER pathway. In this study, we aimed to investigate the associations between glioma risk and XRCC1 Arg399Gln and PARP1 Val762Ala polymorphisms per se and in combination. XRCC1 Arg399Gln and PARP1 Val726Ala polymorphisms were investigated by PCR-RFLP method in 119 glioma patients and 180 cancer-free control subjects. The results were statistically analysed by calculating the odds ratios (OR) and their 95% confidence intervals (95% CI) using the χ2 tests. Glioma patients in this study had significantly higher frequencies of XRCC1 Arg399Gln polymorphism both in homozygote (GG) and heterozygote (AG) status (31% and 56%, respectively) (p < 0.001), and also increased frequency of 399Gln (G) allele (59%) (p < 0.001). Val/Ala (VA) genotype of PARP1 Val762Ala polymorphism was significantly more in the control group (p = 0.02). The combined genotypes of XRCC1 AG or GG with PARP1 VA or AA, and XRCC1 AG or GG with PARP1 VV were more represented in the glioma patients (p = 0.001 and 0.003, respectively). We conclude that XRCC1 Arg399Gln polymorphism is a significant risk factor, and 399Gln (G) allele carries a 3.5 times greater risk for glioma, while PARP1 Val/Ala genotype may be protective against it. We also suspect that in the presence of a polymorphic (G) allele of XRCC1, the plausible protective effect of PARP1 VA genotype may be greatly suppressed.

DNA repair gene XRCC1 polymorphisms and the risk of asthma in a Turkish population.

Polymorphisms have been identified in several DNA damage repair genes. These polymorphisms may effect DNA repair capacity and modulate asthma susceptibility. In this study, we aimed to determine the two polymorphisms in DNA repair gene, x-ray repair cross-complementing group 1 (XRCC1), in a sample of Turkish patients with asthma, and evaluate their association with asthma development. We used polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) to analyze XRCC1 Arg194Trp and XRCC1 Arg399Gln polymorphisms in 116 patients with asthma and in 180 disease-free controls. Our data showed a positive association between the polymorphisms of codons 194 (odds ratio [OR] = 1.97, 95% confidence interval [CI] = 1.06-3.66, and p = 0.03 for Arg/Trp genotype) and 399 (OR = 1.87, 95% CI = 1.12-3.13, and p = 0.02 for Arg/Gln genotype, and OR = 2.59, 95% CI = 1.24-5.43, and p = 0.01 for Gln/Gln genotype) and asthma risk. No statistically significant difference was found for the allelic and genotypic distributions of the polymorphisms in XRCC1 gene between mild and moderate asthmatic patients. A combined analysis of the effect of XRCC1 codons 194 and 399 revealed the highest risk (OR = 4.17, 95% CI = 1.77-9.83, and p = 0.001) for carriers of the polymorphic alleles in both of these codons. These results suggest that the risk of asthma may be associated with DNA repair mechanisms, and understanding these mechanisms will help identify individuals at increased risk of developing asthma and should lead to improved treatment of asthma.

The Ala allele at Val762Ala polymorphism in poly(ADP-ribose) polymerase-1 (PARP-1) gene is associated with a decreased risk of asthma in a Turkish population.

BACKGROUND AND OBJECTIVE: It has been suggested that inhibition of poly (ADP-ribose) polymerase-1 (PARP-1), either pharmacologically or by a gene knockout provides significant protection against systemic or tissue inflammation in animal models. The aim of this study was to analyze the association of the PARP-1 Val762Ala polymorphism, which has beenreported to be associated with decreased enzymatic activity, in Turkish patients with adult asthma. METHODS: A total of 112 subjects with stable asthma and 180 normal controls from the same geographic region were studied and polymerase chain reaction-based restriction analysis was used to identify Val762Ala polymorphism of the PARP-1. RESULTS: In univariate analysis, PARP-1 762 AA genotype conferred a 3.4 fold reduction in risk (OR = 0.297, 95% CI = 0.105-0.813; P = 0.014), while heterozygous VA genotype conferred an even greater level of protection (OR = 0.06; 95%CI, 0.026-0.14; P < 10(-6)). In addition, wild type PARP-1 762 V allele had 5 times the risk of developing asthma than those without the allele (OR 0.199, CI 0.118-0.334, P = 10(-6)). CONCLUSIONS: These findings suggest that PARP-1 V762A variants may be one of the factors participating in protection or susceptibility to asthma in our population.

The Val762Ala polymorphism in the poly(ADP-ribose) polymerase-1 gene is not associated with susceptibility in Turkish rheumatoid arthritis patients.

The findings of the studies on poly(ADP-ribose) polymerase-1 (PARP-1) have suggested that the enzyme inactivation provides significant protection against systemic or tissue inflammation in animal models. It has also shown that the single-nucleotide polymorphism (Val762Ala) of the PARP-1 causes about 40% decrease of enzyme activity. The aim of this study was to analyze the association of the PARP-1 Val762Ala polymorphism in Turkish patients with rheumatoid arthritis. A total of 128 RA patients and 165 normal controls from the same geographic region were studied and polymerase chain reaction (PCR)-based restriction analysis was used to identify Val762Ala polymorphism of the PARP-1. Association analyses were performed using chi (2) tests. Our results indicated that the distribution of the PARP-1 genotypes and alleles did not differ significantly among subjects with or without RA (P > 0.05). The results of the study indicate that, for our Turkish sample, the V762A polymorphism of the PARP-1 may not be involved in susceptibility to RA, implying that the polymorphism may not function as a candidate gene marker for screening RA patients.

Does metformin prevent short-term oxidant-induced dna damage? In vitro study on lymphocytes from aged subjects.

Metformin(1-(diaminomethylidene)-3,3-dimethyl-guanidine), an anti-hyperglycemic agent, also has antioxidant effects. Although the origin is not clearly understood, the antioxidant activity of metformin might result from a direct effect on reactive oxygen species (ROS) or could have an indirect action on the superoxide anions produced by hyperglycemia. The ability of metformin to modulate DNA damage produced by oxidative stress is not known. For this reason, we examined the short term effect of metformin (50 microM, 2 h) on the DNA damage of cumene hydroperoxide (CumOOH)-induced lymphocytes from aged and young control groups (n = 10 each). In this study, DNA damage elicited by CumOOH (1 mM) was detected with the Comet Assay and the ELISA technique. Our results showed a significant increase in apoptotic DNA fragmentation and DNA strand breaks (Comet assay tail factor %) that was detected before and after CumOOH induction in lymphocytes of healthy elderly subjects when compared with healthy young control. Metformin significantly decreased CumOOH-induced apoptotic DNA fragmentation and DNA strand breaks in lymphocytes from aged subjects, although it did not produce a long-term effect. The in vitro results indicate that the short-term effect of metformin can protect against prooxidant stimulus-induced DNA damage in lymphocytes from elderly subjects.