Metformin-treated Group Research Articles

Metformin use is associated with low risk of case fatality and disability rates in first-ever stroke patients with type 2 diabetes.

Background:To assess the effectiveness of metformin treatment on long-term outcomes in first-ever stroke patients with type 2 diabetes mellitus (T2DM) in China.Methods:From August to September 2019, all patients with first-ever stroke and T2DM from 232 hospitals in China Mainland were included. The enrolled patients were divided into two groups: the metformin treatment (MT) and the no-metformin treatment (No-MT) groups. All discharged patients would receive a telephone follow-up at 12-month after admission.Results:In total, 7587 first-ever stroke patients with T2DM [age: median (IQR) = 66 (57–73) years; 57.35% male] were recruited. Out of those 7587 included patients, 3593 (47.36%) received MT. The in-hospital case fatality rate was lower in the MT group than the No-MT group [MT group versus No-MT group: 1.09% versus 2.30%; absolute difference = −1.75% (95% CI = −2.15 to −1.17%); OR = 0.63 (95% CI = 0.47 to 0.84)]. The 12-month case fatality rate was lower in the MT group than the No-MT group [4.72% versus 8.05%; absolute difference = −4.05% (95% CI = −5.58 to −2.41); OR = 0.69 (95% CI = 0.50 to 0.88)]. The 12-month disability rate was also lower in the MT group than the No-MT group [14.74% versus 19.41%; absolute difference = −5.70% (95% CI = −7.25 to −3.22); OR = 0.83 (95% CI = 0.70 to 0.95)]. Furthermore, the recurrence rate did not differ significantly between the MT and No-MT groups (p = 0.29).Conclusion:The study reveals that metformin use in stroke patients with T2DM results in a less severe stroke and lower fatality and disability rates.

The Effect of Endometrial Cell Culture on α3 and β1 integrin Genes and Protein Expression in Type 2 Diabetic Rats at The Time of Implantation.

Objective Given the prevalence of fertility problems in couples and the defect in embryo implantation as well as the low success rate of assisted reproductive techniques, it is necessary to investigate the causes of this phenomenon. Type 2 diabetes mellitus (T2DM) is a metabolic disease with multiple effects on various organs as well as the endometrium. In this study, the effects of endometrial cell culture on the expression of α3 and β1 integrin genes and protein in type 2 diabetic rats were investigated. Materials and Methods In this experimental study, 35 female rats were divided into five groups: control, sham, diabetic, Pioglitazone-treated and Metformin-treated groups. First, rats were maintained in diabetic condition for 4 weeks. Then, treatment was performed for the next four weeks. Four weeks after induction of diabetes, rats were sacrificed at the time of embryo implantation. The uterus was removed. Endometrial cells were isolated and cultured for 7 days. Immunocytochemistry staining was used to confirm endometrial cells. Expression of α3 and β1 integrin genes was determined by real-time polymerase chain reaction (PCR) technique and the α3β1 protein content measured using Western blot both before and after endometrial cell culture.ResultsThe expression level of α3 integrin gene in the Pioglitazone-treated group compared with metformin-treated group was significantly decreased (P<0.001). The same result was observed in β1 integrin gene expression (P=0.004). Also, the α3β1 protein level increased in all diabetic groups, but its reduction was significantly greater in pioglitazone- treated group (P=0.004). ConclusionT2DM altered the expression of α3 and β1 integrin genes and related proteins, which endometrial cell culture regulated this disorder. According to these results, may be the endometrial cell culture can reduce the adverse effects of diabetes on α3 and β1 integrin expression at the level of gene and protein, in endometrial cells.

Comparison of Effect of Metformin Versus Combination of Folic Acid/Myo-inositol in Infertile Women with Poly Cystic Ovary Syndrome Undergoing In Vitro Fertilization: A Randomized Clinical Trial

Background: Previous studies have reported contradictory results regarding the effectiveness of metformin and myo-inositol on improving reproductive outcomes in patients with polycystic ovary syndrome (PCOS). Therefore, this study aimed to compare the effectiveness of these two drugs on improving reproductive outcomes of PCOS patients undergoing in vitro fertilization (IVF) treatment. Methods: This double-blind randomized clinical trial study was con­ducted on 140 infertile women with PCOS who were scheduled to undergo IVF in Yas Hospital, Tehran, Iran in 2020. The patients were randomly divided into two groups, including metformin and combination of myo-inositol and folic acid treatment groups. Reproductive outcomes were compared between the two groups. Results: The mean age of the patients was 30.66 ± 5.7 years (30.51 ± 5.88 in metformin treatment group and 30.9 ± 5.53 in myo-inositol group, P = 0.68). There was no significant difference between the two groups regarding the reproductive outcomes, including pregnancy rate, change in BMI, number and quality of oocytes, number and quality of embryos, follicle number, follicle size, ectopic pregnancy rate, and abortion rate (P &gt; 0.05). Conclusion: There is no significant difference in the effect of metformin and myo-inositol on improving reproductive outcomes. Therefore, due to the lack of significant differences in the effectiveness of these two drugs and considering their availability and cost, one of them can be prescribed for the patients.

Salvia spinosa L. Protects against Diabetes-Induced Nephropathy by Attenuation of Mitochondrial Oxidative Damage in Mice.

Mitochondrial oxidative damage is a crucial factor in the pathogenesis of diabetic nephropathy (DN), which is among the most prevalent problems of diabetes, and there hasn't been an effective treatment for DN yet. This study planned to investigate the effects of Salvia spinosa L. on mitochondrial function along with its protection against streptozotocin-induced nephropathy in diabetic mice. After the injection of streptozotocin (STZ) and verification of the establishment of diabetes, mice (n = 30) were randomly divided into the following groups: control group, diabetic-control, S. spinosa-treated diabetic (50, 100, and 200 mg/kg), and metformin-treated diabetic group (500 mg/kg). After four weeks of treatment, the mice were weighed. Blood and kidney tissues were examined for biochemical and histological evaluation. Hematoxylin and eosin staining was used for evaluating renal pathologic damage. Oxidative damage in the kidney was assessed by the evaluation of lipid peroxidation and glutathione oxidation. Furthermore, differential centrifugation was used to obtain the isolated mitochondria, and mitochondrial toxicity endpoints (mitochondrial function and mitochondrial oxidative markers) were determined in them. S. spinosa remarkably reduced the blood urea and creatinine concentrations, and also normalized kidney weight/body weight coefficient in the diabetic mice. S. spinosa ameliorated the incidence of glomerular and tubular pathological changes in histological analyses. Moreover, the oxidative and mitochondrial damages were notably attenuated in renal tissues of S. spinosa-treated mice. These results indicate that the methanolic extract of S. spinosa modulates the nephropathy in the diabetic mice by the amelioration of oxidatively induced mitochondrial damage and provides a reliable scientific base, suggesting S. spinosa as a promising alternative remedy against DN.

Effects of orlistat and metformin on metabolism and gonadal function in overweight or obese patients with polycystic ovary syndrome

The aim of this study was to assess the effects of orlistat or metformin treatment on lipid and glucose metabolism, and gonadal function in obese/overweight women with polycystic ovary syndrome (PCOS). A total of 39 patients diagnosed with PCOS were randomly (digital table method) divided into orlistat treatment group (20 cases) and metformin treatment group (19 cases). Compared with those before, treatment with either orlistat or metformin significantly reduced body weight, body mass index (BMI), hip circumferences, and serum insulin levels of the PCOS patients both at the end of 3 months and 6 months (P<0.05). No significant differences could be viewed between orlistat and metformin treated subjects. Moreover, orlistat treatment significantly lowered the levels of low-density lipoprotein cholesterol, total cholesterol, fasting blood glucose, and homeostasis model assessment-insulin resistance (HOMA-IR) (P<0.05), while there were no significant changes in above parameters with metformin treatment. The improvement of menstrual cycle was observed after 6-month treatment in both groups (P<0.05). However, changes in polycystic ovarian morphology by ultrasound were only observed in orlistat treated group. In conclusion, orlistat is comparable with metformin in weight loss and improvement of insulin resistance and menstrual cycle, and is superior to metformin in improvement of lipid metabolism in overweight/obese PCOS patients.

Metformin enhances the osteogenesis and angiogenesis of human umbilical cord mesenchymal stem cells for tissue regeneration engineering.

Human umbilical cord mesenchymal stem cells (hUC-MSCs) are a potential clinical material in regenerative medicine applications. Metformin has shown safety and effectiveness as a clinical drug. However, the effect of metformin as a treatment on hUC-MSCs is unclear. Our research aimed to explore the effects of metformin on the osteogenesis, adipogenesis and angiogenesis of hUC-MSCs, and attempted to explain the molecular fluctuations of metformin through the mapping of protein profiles. Proliferation assay, osteogenic and adipogenic differentiation induction, cell cycle, flow cytometry, quantitative proteomics techniques and bioinformatics analysis were used to detect the influences of metformin treatment on hUC-MSCs. Our results demonstrated that low concentrations of metformin promoted the proliferation of hUC-MSCs, but high concentrations of metformin inhibited it. Metformin exhibited promotion of osteogenesis but inhibition of adipogenesis. Metformin treated hUC-MSCs up-regulated the expression of osteogenic marker ALP, OCN and RUNX2, but down-regulated the expression of adipogenic markers PPARγ and LPL. Proteomics analysis found that up-regulation of differentially expressed proteins in metformin treatment group involved the biological process of cell migration in Gene Ontology analysis. Metformin enhanced cell migration of HUVEC in a co-culture system, and hUC-MSCs treated with metformin exhibited stronger angiogenesis in vitro and in vivo compared to the hUC-MSCs group. The results of RT-qPCR revealed that the SCF and VEGFR2 were raised in metformin treatment. This study can promote the application of hUC-MSCs treated with metformin to tissue engineering for vascular reconstruction and angiogenesis.

Moringa oleifera Lam. seed extract protects kidney function in rats with diabetic nephropathy by increasing GSK-3β activity and activating the Nrf2/HO-1 pathway

BackgroundMoringa oleifera Lam. (M. oleifera) seeds are widely used in traditional folk medicine and as nutritional supplements in the Middle East, Africa, and other regions. Published research showed that M. oleifera seeds (MOS) have pharmacological activities such as blood glucose-lowering, anti-inflammatory, and antitumor effects. However, experimental evidence on the use of MOS to treat diabetic nephropathy and its underlying mechanisms were rarely reported. PurposeTo evaluate the therapeutic effects of MOS extract on the kidneys of high-fat diet (HFD)-fed rats with streptozotocin-induced diabetic nephropathy and reveal its underlying mechanisms. Study designHFD-fed rats with streptozotocin-induced diabetic nephropathy and high-glucose induced Human Renal Mesangial Cells (HRMC) were used to explore the protective effect of MOS on diabetic nephropathy in vivo and in vitro. Methods: HRMC were used to preliminarily evaluate the effect of MOS extract under high glucose conditions. For the in vivo study, rats were divided into the following 6 groups (n = 5): normal control group (NC), diabetic nephropathy model group (DN), high dose of MOS-treatment group (DN + MOS-H, 200 mg/kg/d); medium dose of MOS-treatment group (DN + MOS-M, 100 mg/kg/d); low dose of MOS-treatment group (DN + MOS-l, 50 mg/kg/d), and metformin-treatment group (DN + MET, 200 mg/kg/d). After 4 weeks of treatment, the damage caused by DN was assessed based on the related parameters of urine and blood. Periodic acid-Schiff (PAS) staining and hematoxylin and eosin (HE) staining were used to assess pathological tissue damage. Immunohistochemistry was used to detect nuclear factor erythroid-derived 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and phosphorylated-glycogen synthase kinase-3beta (P-GSK-3β) levels, whereas western blotting was used to detect Nrf2, HO-1, nephrin, GSK-3β, and p-GSK-3β levels. ResultsMOS extract could inhibit the proliferation of HRMCs induced by high glucose levels. Compared with the rats in the DN group, MOS not only significantly reduced blood glucose levels and oxidative stress in the experimental rats but also improved their kidney function and reduced kidney tissue damage. Additionally, MOS extract increased GSK-3β activity and the expression of Nrf2 and HO-1. ConclusionsThis study showed that MOS could activate GSK-3β and Nrf2/HO-1 pathways to exert antioxidant and anti-renal fibrosis activities, and delay the progression of diabetic nephropathy.

UHPLC-MS-Based Serum and Urine Metabolomics Reveals the Anti-Diabetic Mechanism of Ginsenoside Re in Type 2 Diabetic Rats.

Panax ginseng was employed in the treatment of “Xiao-Ke” symptom, which nowadays known as diabetes mellitus, in traditional Chinese medicine for more than a thousand years. Ginsenoside Re was the major pharmacologic ingredient found abundantly in ginseng. However, the anti-diabetic of Ginsenoside Re and its underlying mechanism in metabolic level are still unclear. Serum and urine metabolomic method was carried out to investigate the anti-diabetic pharmacological effects and the potential mechanism of Ginsenoside Re on high-fat diet combined streptozotocin-induced type 2 diabetes mellitus (T2DM) rats based on ultra-high-performance liquid chromatography coupled with quadrupole exactive orbitrap mass spectrometry (UHPLC-Q-Exactive Orbitrap/MS). Serum and urine samples were collected from the control group (CON), T2DM group, metformin (MET) treatment group, and ginsenoside Re treatment group after intervention. The biochemical parameters of serum were firstly analyzed. The endogenous metabolites in serum and urine were detected by UHPLC-MS. The potential metabolites were screened by multivariate statistical analysis and identified by accurate mass measurement, MS/MS, and metabolite databases. The anti-diabetic-related metabolites were analyzed by KEGG metabolic pathway, and its potential mechanism was discussed. The treatment of ginsenoside Re significantly reduced the blood glucose and serum lipid level improved the oxidative stress caused by T2DM. Biochemical parameters (urea nitrogen, uric acid) showed that ginsenoside Re could improve renal function in T2DM rats. Respective 2 and 6 differential metabolites were found and identified in serum and urine of ginsenoside Re compared with T2DM group and enriched in KEGG pathway. Metabolic pathways analysis indicated that the differential metabolites related to T2DM were mainly involved in arachidonic acid metabolism, Vitamin B6, steroid hormone biosynthesis, and bile secretion metabolic pathways. This study verified the anti-diabetic and anti-oxidation effects of ginsenoside Re, elaborated that ginsenoside Re has a good regulation of the metabolic disorder in T2DM rats, which could promote insulin secretion, stimulated cannabinoid type 1 receptor (CB1), and CaMKK β to activate AMPK signaling pathway, inhibited insulin resistance, and improved blood glucose uptake and diabetic nephropathy, so as to play the role of anti-diabetic.

Effect of hydrogen-rich water on the lactic acid level in metformin-treated diabetic rats under hypoxia.

The present study aims to investigate the impact of hydrogen-rich water on the lactic acid level in metformin-treated diabetic rats under hypoxia. Thirty Sprague-Dawley rats were randomly divided into five groups, including normal diet group, and diabetes model (DM) group, DM + metformin treatment (DMM) group, DMM + hypoxia treatment (DMMH) group and DMMH + hydrogen-rich water (DMMHR) group. We found that the levels of lactic acid, pyruvate and lactate dehydrogenase were significantly lower in the blood of DMMHR group than DMMH group. Superoxide dismutase and glutathione levels in liver and heart were significantly higher in DMMH group after hydrogen-rich water treatment, while malondialdehyde and oxidized glutathione levels were decreased in DMMHR group when compared with DMMH group, which indicates that hydrogen-rich water could reduce oxidative stress. qPCR analysis demonstrated that that pro-apoptotic genes Bax/Caspase-3 were upregulated in DM group and metformin treatment suppressed their upregulation (DMM group). However, hypoxic condition reversed the effect of metformin on apoptotic gene expression, and hydrogen-rich water showed little effect on these genes under hypoxia. HE staining showed that hydrogen-rich water prevented myocardial fiber damages under hypoxia. In summary, we conclude that hydrogen-rich water could prevent lactate accumulation and reduce oxidant stress in diabetic rat model to prevent hypoxia-induced damages. It could be served as a potential agent for diabetes patients with metformin treatment to prevent lactic acidosis and reduce myocardial damages under hypoxic conditions.

Modulatory Effects of Metformin Alone and in Combination with Cimetidine and Ibuprofen on T Cell-related Parameters in a Breast Cancer Model.

Metformin, cimetidine, and ibuprofen separately exhibit immunomodulatory and anti-tumorigenic effects. Herein, the impacts of metformin alone and in combination with cimetidine/ibuprofen on some Th1- and regulatory T (Treg) cell-related parameters were evaluated using a breast cancer (BC) model. For establishing the BC model, four groups of Balb/c mice were challenged with the carcinoma cell line. After 11-30 days post-induction, they were treated intraperitoneally (with metformin (200 mg/kg), "metformin plus cimetidine (20 mg/kg)"; "metformin plus ibuprofen (20 mg/kg)", or with all three drugs in mentioned doses. Untreated BC and without tumor mice were enrolled as control groups. On day 31, splenic Th1 and Treg cell frequencies, serum interferon-gamma (IFN-γ), and transforming growth factor-beta (TGF-β) concentration, and intra-tumoral T-bet, TGF-β, and forkhead box protein P3 (FOXP3) expression were measured; using flow cytometry, enzyme-linked immunosorbent assay (ELISA), and real-time-PCR, respectively. Treatment of the BC mice with metformin alone and in combination with cimetidine and/or ibuprofen enhanced the frequency of Th1 cells, and IFN-γ concentration, while it resulted in a decrease in the frequency of Treg cells, serum TGF-β concentration, and the expression of FOXP3 and TGF-β compared with un-treated BC mice. FOXP3 expression in the metformin-treated group was lower in mice who received combination therapy. Survival rate and body weight were increased, while tumor size and spleen index were reduced in mice treated with metformin alone and its combination with cimetidine and/or ibuprofen. No remarkable differences were found between metformin-treated mice and those who received combination therapies regarding Th1 and Treg cell percentages, TGF-β expression, body weight, tumor size, and spleen index. The benefits of combinational therapy may be largely attributed to metformin. Immunotherapeutic potentials of metformin in cancers need further considerations.

The Effectiveness of Traditional Chinese Medicine Jinlida Granules on Glycemic Variability in Newly Diagnosed Type 2 Diabetes: A Double-Blinded, Randomized Trial.

This study aimed to evaluate the influence of Jinlida granules on glycemic variability with or without metformin treatment in patients with newly diagnosed type 2 diabetes. This study was a 16-week, double-blinded, randomized, controlled clinical trial. The enrolled patients with newly diagnosed type 2 diabetes were randomly divided into four groups: control, Jinlida, metformin, and combination treatment groups. A retrospective continuous glucose monitoring (CGM) system was used for subcutaneous interstitial glucose monitoring for 3 days consecutively. Hemoglobin A1c (HbA1c), traditional Chinese medicine symptom score, and CGM parameters, including glucose coefficient of variation, standard deviation of blood glucose values, and time in range of glucose 3.9–10.0 mmol/L, were assessed pre-test and post-test. A total of 138 participants completed the entire procedure. Compared with the pre-test, fasting plasma glucose, 2 hour postprandial plasma glucose, HbA1c, and traditional Chinese medicine symptom score all decreased in the four groups at the end of the test, and the combination treatment group showed the most significant decrease. In terms of CGM parameters, time in range of the Jinlida and metformin groups improved after intervention compared with the baseline (Jinlida group: 78.68 ± 26.15 versus 55.47 ± 33.29; metformin group: 87.29 ± 12.21 vs. 75.44 ± 25.42; P < 0.01). Additionally, only the Jinlida group showed decreased glucose standard deviation after intervention (1.57 ± 0.61 vs. 1.96 ± 0.95; P < 0.01). Jinlida granules can improve glycemic control and glycemic variability in patients with newly diagnosed type 2 diabetes. Clinical trial registration number: ChiCTR-IOR-16009296.

Metformin promotes autophagy activity and constrains HSV-1 replication in neuroblastoma cells

Problem consideredMetformin is an antihyperglycemic drug with effective clinical functions in type 2 diabetes patients. Recent evidence indicates that metformin can change natural cellular signaling to induce autophagy. Furthermore, infection could rise autophagy activity, but herpes simplex virus 1 (HSV-1) constructs, particularly virulence factors, constrain autophagy promotion, we used HSV-1 because of its ability to escape from autophagy by inhibiting beclin-1 formation, reversing PKR signaling and also induce mTOR activity. This study aimed to compare the extent of autophagic activity in metformin-treated cells infected with HSV-1 and determine the effect of metformin on HSV-1 replication. MethodsNeuroblastoma cells were cultured and then divided into the two separate groups of infected with HSV-1 and expose with metformin. Autophagy was evaluated via microtubule-associated protein 1 light chain 3 (LC3-II) monitoring by affix staining antibodies to this protein using flow cytometry. To evaluate viral load, HSV-1 was inoculated for 4 h post metformin treatment, and to evaluate viral load in the post-infection pathway, cells were inoculated with HSV-1. Then, after a 2-hour viral attachment time, metformin was administered to the cells. Viral nucleic acid was purified 48 h post-infection, and virus titer was calculated by real-time polymerase chain reaction (PCR). ResultsOur real-time PCR results indicated that HSV-1 viral load in pre-infection pathway reduced from 1.3 × 106 in positive control to 8 × 105 in cells exposed to metformin. Additionally, the TCID50/ml at 12 and 24 h after infection was 10 times lower than in HSV-1 positive control. However, after 48 h, HSV-1 titer in the metformin-treated group was equal with non-treated cells. For the post-infection pathway, real-time PCR results exhibited that HSV-1 titer was 2.3 × 106 in virus control cells compared to 1.8 × 106 in treated cells. That is, TCID50/ml in the medium of metformin-treated cells was 10 times lower in comparison with non-treated cells after 72 h. ConclusionThe study of the effect of metformin on neuroblastoma cells indicates high autophagic flux and lesser HSV-1 propagation in treated cells.

Metformin Exerts Anti-inflammatory and Mucus Barrier Protective Effects by Enriching Akkermansia muciniphila in Mice With Ulcerative Colitis.

The present study aimed to determine if metformin exerts anti-inflammatory and mucus-protective effects via the gut microbiota. Metformin has extensive benefits including anti-inflammatory effects. Previous studies showed that metformin changed the gut microbiota composition and increases the number of goblet cells. Intestinal dysbiosis and goblet cell depletion are important features of ulcerative colitis (UC). The underlying mechanism and whether metformin can improve the mucus barrier in UC remain unclear. Metformin (400 mg/kg/day) was administered to mice with dextran sulfate sodium (DSS)-induced UC for 2 wk to investigate the effects of metformin on the intestinal mucus barrier. The gut microbiota was depleted, using antibiotics, to explore its role in the mucus-protecting effects of metformin. Akkermansia muciniphila (A. muciniphila), which was enriched in metformin-treated mice, was administered to mice to investigate the effects of the bacteria on UC and the mucus barrier. Metformin attenuated DSS-induced UC in mice, as evidenced by the alleviation of diarrhea, hematochezia, and the decrease in body weight. The expression of mucin2, a prominent mucus barrier protein, was increased in the metformin-treated group compared to the DSS-treated group. Furthermore, fecal 16S rRNA analysis showed that metformin treatment changed the gut microbiota composition by increasing the relative abundance of Lactobacillus and Akkermansia species while decreasing Erysipelatoclostridium at the genus level. Antibiotic treatment partly abolished the anti-inflammatory and mucus-protecting effects of metformin. Administration of A. muciniphila alleviated the colonic inflammation and mucus barrier disruption. Metformin alleviated DSS-induced UC in mice and protected against cell damage via affecting the gut microbiota, thereby providing a new mechanism for the therapeutic effect of metformin in patients with UC. This study also provides evidence that A. muciniphila as a probiotic has potential benefits for UC.

Histological investigation of the protective effect of metformin on testis and sperm parameters in obese rats with type 2 diabetes mellitus

Background/Aim: Diabetes mellitus (DM) is thought to have adverse effects on the male reproductive system. Metformin (MTF) is a widely used drug in the treatment of DM. This study hypothesized that MTF can reduce the harmful effects of DM on the male reproductive system in addition to diabetes treatment. Methods: Twenty-one adult (15-18 g, 11-12 months) C57BL6 male mice were randomly assigned to three groups, as follows: The Control group (C), Diabetes group (D), and Diabetes + Metformin (D+MTF) treatment group. Groups D and D+MTF were fed a diet composed of 60% fat for four weeks. DM was created by administering a single dose of 30 mg/kg streptozotocin intraperitoneally (i.p.). MTF was given through gavage at a dose of 300 mg/kg/day. At the end of the experiment, sperm parameters were evaluated in the testicular tissue. Histomorphology features and immune expressions of VEGF, Caspase-3, and Ki-67 were evaluated in testicular tissue sections stained with Hematoxylin and Eosin (HE). Results: Sperm concentration, motility, and morphological characteristics of diabetic mice were significantly reduced compared to control, and MTF-treated mice. Seminiferous tubule diameters and Johnsen scores were significantly higher in the control and D+MTF groups when compared to the diabetic group. VEGF and Ki-67 immune expression were significantly higher in the control and D+MTF groups compared to the D group (P=0.001, and P=0.002, respectively). Because of D, Caspase-3 positive cell density was higher in the D group compared to the control and D+MTF groups (P=0.002). Conclusion: Diabetes mellitus had adverse effects on sperm parameters and the testicular tissue. High glucose levels with decreased VEGF immune expression damaged spermatogenic cells and decreased proliferation and differentiation due to impaired intratesticular vascularity. MTF treatment reduced the damage in spermatogenic cells and contributed to the differentiation and division of cells through the restoration of vascularization.

Dynamic switch of immunity and antitumor effects of metformin in rat spontaneous esophageal carcinogenesis

Chronic inflammation contributes to tumor development by creating a local microenvironment that facilitates neoplastic transformation and potentiates the progression of cancer. Esophageal cancer (EC) is an inflammation-associated malignancy with a poor prognosis. The nature of the switch between chronic inflammation of the esophagus and EC-related immunological changes remains unclear. Here, we examined the dynamic alterations of immune cells at different stages of chronic esophagitis, Barrett’s esophagus (BE) and EC using an esophageal spontaneous carcinogenesis rat model. We also investigated the anticancer effects of metformin. To stimulate EC carcinogenesis, chronic gastroduodenal reflux esophagitis via esophagojejunostomy was induced in 120 rats in metformin-treated and non-treated (control) groups. After 40 weeks, BE and EC developed in 96.7% and 63.3% of the control group, and in 66.7% and 23.3% of the metformin-treated group, respectively. Flow cytometric analysis demonstrated that the balance of M1/M2-polarized or phospho-Stat3-positive macrophages, regulatory T, cytotoxic T, natural killer (NK), NK T cells, and Th17 T cells was dynamically changed at each stage of the disease and were resolved by metformin treatment. These findings clarify the immunity in esophageal carcinogenesis and suggest that metformin could suppress this disease by improving the immunosuppressive tumor microenvironment and immune evasion.

RETRACTED: Evaluation of the effects of metformin as adenosine monophosphate-activated protein kinase activator on spatial learning and memory in a rat model of multiple sclerosis disease

In patients with multiple sclerosis (MS) disease, cognitive deficits have been detected because of destruction of hippocampus. Cognitive impairment is one of the common signs in MS. Recent studies showed that metformin (Met) has wide-ranging effects in the treatment of diseases. Here, we have tried to study the preservative effects of Met as adenosine monophosphate-activated protein kinase (AMPK) activator on the hippocampus dentate gyrus (DG) neuronal firing pattern, motor coordination, and learning & memory loss following MS induction. The MS induction was done by local ethidium bromide (EB) injection into the rat hippocampus. Then, rats were treated with Met (200 mg/kg) for two weeks. Spatial memory and learning status were assessed using Morris water maze. A neuronal single-unit recording was measured from hippocampus DG. After decapitation, the bilateral hippocampi separated to measure malondialdehyde (MDA). Treatment with Met ameliorated latency times and path lengths (P < 0.05, P < 0.01, P < 0.001 in 1th, 2th, 3th and 4th days) in the Met + MS group respectively. The percent of total time spent in goal quarter and the average number of spikes/bin were decreased significantly in MS rats compared with the sham group (p < 0.001) but significantly increased in the metformin-treated MS group (Met + MS), (p < 0.01, p < 0.001). Met treatment in rats with MS significantly reduced the concentration of MDA, which is an indicator of lipid peroxidation compared to untreated groups. These observations show that increase of neuronal activity, sensory-motor coordination, and improvement of spatial memory in MS rats treated with Met appears via an increment of AMPK.

Metformin treatment confers protection of the optic nerve following photoreceptor degeneration.

Acquired or inherited or photoreceptor loss causes retinal ganglion cell loss and ultimately axonal transport alteration. Thus, therapies should be applied early during photoreceptors degeneration before the remodeling process reaches the inner retina. This study aimed to evaluate the protective effect of metformin on the rat optic nerve following photoreceptors loss induced by N-Ethyl-N-nitrosourea (ENU). Eighteen adults male Wistar rats were divided into two groups. Group I: normal vehicle control (n=6). Group II: ENU-induced photoreceptors degeneration (n=12) received a single intraperitoneal injection of ENU at a dose of 600 mg/kg. Rats in group II were equally divided into two subgroups: IIa: photoreceptor degeneration induced group and IIb: metformin treated group (200 mg/kg) for 7 days. Specimens from the optic nerve were processed for light and electron microscopy. In ENU treated group, the optic nerve revealed reduction in the diameter of the optic nerve fibers and thinning of myelin sheath with morphological changes in the glia (astrocytes, oligodendrocytes, and microglia). Caspase-3 (apoptotic marker), iNOS (oxidative stress marker) and CD68 (macrophage marker) expression increased. In metformin-treated group, the diameter of optic nerve fibers and myelin sheath thickness increased with improvement of the deterioration in the glia. Caspase-3, iNOS and CD68 expression decreased. Metformin ameliorates the histological changes of the rat optic nerve following photoreceptors loss induced by ENU.

Metformin Reduces Tumor Burden and Splenomegaly in JAK2V617F Murine Models

Introduction : The recurrent gain-of-function JAK2V617F mutation confers growth factor-independent proliferation for hematopoietic cells and is a major contributor to the pathogenesis of myeloproliferative neoplasms (MPN). Metformin (1,1-dimethylbiguanide) is a biguanide widely prescribed for the treatment of type II diabetes and metabolic syndromes. In recent years, studies using cancer cell lines and murine models have provided evidence for potential anticancer activity of metformin. Previous results from our research group indicate that metformin reduces cell viability, proliferation, clonogenicity and JAK2/STAT activation in human JAK2V617F cell lines and spontaneous erythroid colony formation in primary cells from patients with polycythemia vera (Machado-Neto et al. ASH Annual Meeting Abstracts #1960, 2016). Objectives : To investigate the effects of metformin treatment in tumor formation and MPN phenotype using Ba/F3 JAK2V617F cells and Jak2V617F knockin-induced MPN mice. Methods : For tumor formation, experimental groups consisted of 8-10 week-old NSG female mice that received a 100 µL (50 µL PBS plus 50 µL matrigel) dorsal subcutaneous injection of 2×106 Ba/F3 JAK2V617F cells and daily treatment by intraperitoneal injection of vehicle (PBS, n=9) or metformin (125 mg/kg, n=9) for 13 days after initial tumor growth (7 days). Tumor volume (V) was obtained using the formula (V = W2 × L × 0.52), where W and L are the smaller and larger diameters, respectively. For MPN phenotype induction, 5×106 bone marrow cells from Jak2V617F knockin (Jak2WT/V617F) mice were transplanted into lethally irradiated Pep boy mice. After 4 weeks, chimerism was evaluated by CD45.1 and CD45.2 markers (Becton-Dickinson) by flow cytrometry in peripheral blood. Mice with >70% CD45.2 cells were randomized and treated daily by intraperitoneal injection of vehicle (PBS, n=4) or metformin (125 mg/kg, n=5) for 6 weeks. At the conclusion of the experiment, animals were harvested and subjected to analysis of spleen and hematological parameters. Erythroid progenitors in the spleen were evaluated by CD71 and Ter119 markers (Becton-Dickinson) by flow cytometry. All experiments were approved by the Animal Ethics Committee. For comparisons, Mann-Whitney test or ANOVA test and Bonferroni post-test was used. A p value <0.05 was considered as statistically significant. Results : In NSG mice inoculated with Ba/F3 JAK2V617F, metformin treatment (125 mg/kg/day) was able to significantly reduce the tumor burden after 13 days of treatment. At the endpoint, the mean±SD of tumor volume for vehicle (n=4) and metformin (n=4) treated mice was 1120.7±289.1 mm3 and 510.3±244.4 mm3, respectively (p <0.001). A second independent experiment including five mice in each group presented similar results (vehicle versus metformin: 1307.7±511.9 mm3versus 725.1±380.6 mm3; p <0.01). In Jak2V617F knockin-induced MPN animals, metformin treatment (125 mg/kg/day) was well-tolerated, reduced splenomegaly (p=0.02) and improved splenic architecture. Erythoid progenitors in the spleen and bone marrow did not differ between control and metformin-treated groups (p >0.05), but the smaller spleens presented with a very low percentage of erythroid progenitors. Hemoglobin and hematocrit were not modulated by metformin treatment. Conclusions : Metformin treatment at a dose of 125 mg/kg/day effectively reduced tumor burden in Ba/F3 JAK2V617F allograft and splenomegaly in Jak2V617F knockin-induced MPN murine models. In humans, this dose represents a pharmacological dose similar to that which is used for type II diabetes treatment. Our results provide additional insights for metformin in preclinical models of MPN. DisclosuresDruker:Millipore: Patents & Royalties: Royalties from Dana-Farber Cancer Institute, which has an exclusive commercial license with Millipore for monoclonal antiphosphotyrosine antibody 4G10, which I developed while employed at DFCI.; Henry Stewart Talks: Patents & Royalties; Novartis: Research Funding; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties: #843 Mutated ABL Kinase Domains (licensed to various companies); #0996 Detection of Gleevec Resistant Mutations (licensed to various companies, including MolecularMD); #0606 Treatment of Gastrointestinal Stromal Tumors (exclusively licensed to Novartis); Beta Cat: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Baxalta US Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; McGraw Hill: Patents & Royalties; ARIAD: Research Funding; Aptose Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; MED-C: Membership on an entity's Board of Directors or advisory committees; GRAIL: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; The Leukemia & Lymphoma Society: Other: Joint Steering Committee of AML Master Protocol, Research Funding; Third Coast Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche TCRC: Consultancy, Membership on an entity's Board of Directors or advisory committees; CTI Biopharma: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Cylene: Consultancy, Membership on an entity's Board of Directors or advisory committees; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Monojul: Consultancy; Bristol-Myers Squibb: Research Funding.

The impact of cichorium intybus L. On GDF-15 level in obese diabetic albino mice as compared with metformin effect.

Diabetes mellitus (DM) and obesity comorbidity signify a frequent metabolic disorder, representing a huge public health burden. Metformin, the most used anti-diabetic medication, is found to reduce body weight via growth differentiation factor 15 (GDF-15) signalling pathways. The medicinal herb Cichorium intybus L. (chicory or cichorium) has a promising pharmacological impact on energy homeostasis. On the other hands, little data is available on its role in DM and obesity. Despite its irrefutable effect, its exact mechanism of action has not completely elucidated; the present study evaluated the effect of chicory on DM, antioxidant status, inflammation, and GDF-15 level in comparison with the metformin effect. Eighty albino mice were grouped as (control, obese diabetic group, metformin-treated, and Cichorium intybus L. -treated group). The study assessed blood glucose, lipid profile, inflammatory markers (IL-6, TNF-α), total antioxidant capacity (TAC) and caspase-3. Quantitative RT-PCR assessed GDF-15 and leptin relative mRNA expression. Cichorium intybus L. has significantly lowered inflammatory, apoptotic markers, and leptin levels compared with the diseased group. Likewise, the plant upregulated GDF-15 and TAC's levels. The study documented a non-significant difference between the Cichorium intybus L. -treated and the metformin-treated groups in all estimated markers. The Cichorium intybus L. is a promising herbal supplement with anti-inflammatory, antioxidant, anti-diabetic, and weight reduction effects via affecting GDF-15 signalling pathways. GDF-15 has anti-inflammatory, anti-oxidative stress and anti-apoptotic effect in DM and obesity via targeting NF-κB mechanisms.

Spexin and Metformin Comparative Ameliorated Ovarian and Liver Function Changes in Letrozole-Induced Polycystic Ovary Syndrome in Rats (Histological, Biochemical, Immunohistochemical and Morphometric study)

Background: Polycystic ovary syndrome (PCOS) is characterized by polycystic ovarian follicle morphology, ovulatory dysfunction, hyperandrogenism and insulin resistance. Spexin has a role in satiety control, lipid and glucose metabolism. Metformin is an insulin sensitizing agent that has some hepato-protective functions. Aim: This study aimed to declare and compare the effect of spexin and metformin treatment on ovarian and liver function changes in letrozole-induced PCOS in rats and the possible mechanisms involved.Methods: Thirty-two adult female Wistar albino rats divided into control, polycystic ovary syndrome (PCOS), PCOS metformin-treated, and PCOS spexin-treated groups. In PCOS groups, rats were given oral letrozole 0.5 mg/kg dissolved in 0.9% NaCl once daily along the study. In PCOS metformin-treated group, rats were treated with metformin hydrochloride orally (300 mg/kg/day) for the last 4 weeks. In PCOS spexin-treated group, rats were treated with spexin i.p. 35 μg/kg/day, dissolved in normal saline for the last 4 weeks. Results: In PCOS group, there was a significant increase in; BMI, relative ovarian weight, HOMA-IR, serum (LH, FSH, testosterone, insulin, glucose, TC, TG, LDL, ALT, AST, alkaline phosphatase, total & direct bilirubin) and hepatic (MDA, NO & TNF-), with a significant decrease in; serum [estradiol, progesterone, HDL, albumin and spexin] and hepatic GPX, when compared with the control group. Histopathological results showed ovaries of PCOS marked expression of caspase-3 and P53 and decreased KI67 expression of granulosa and theca cells. Vacuolated hepatocytes, dilated and congested portal vein and positive reaction of immunostaining with αSMA, caspase and TNFα. Treatment of PCOS rats with either metformin or spexin reversed these changes significantly.Conclusion: Spexin serum level could be used as a metabolic biomarker for polycystic ovary syndrome. Also, treatment with either metformin or spexin ameliorated ovarian and hepatic dysfunctions in rat model of PCOS through their insulin sensitizing, anti-oxidant and anti-inflammatory effects.