Different Concentrations Of Metformin Research Articles

Metformin promotes the normalization of abnormal blood vessels after radiofrequency ablation deficiency in hepatocellular carcinoma by microRNA-302b-3p targeting thioredoxin-interacting protein

Metformin has shown great promise in the treatment of HCC. Radiofrequency ablation (RFA) deficiency results in recurrence and metastasis of remaining HCC tumors. Here, we aimed to investigate the role and mechanism of metformin in HCC after RFA deficiency. HCC cell line Hep-G2 was selected to simulate RFA deficiency and named HepG2-H cells. After treating cells with different concentrations of metformin (2.5, 5, 10 μM) or transfecting related plasmids, cell proliferation, migration, invasion, apoptosis and angiogenesis were detected, in vitro permeability test was performed, and an angiogenesis-related protein VEGFA was analyzed. The residual HCC model after RFA deficiency was established in mice. Metformin was administered by gavage to detect changes in tumor volume and weight, and CD31 staining was used to observe microvessels. The targeting relationship between miR-302b-3p and TXNIP was demonstrated by the bioinformatics website, dual-luciferase reporter assay, and RNA pull-down assay. The results found that metformin inhibited RFA deficiency-induced growth and angiogenesis of HCC cells in vitro. miR-302b-3p counteracted the therapeutic effect of metformin on RFA deficiency. miR-302b-3p targeted regulation of TXNIP. The up-regulation of TXNIP reversed the effects of overexpression of miR-302b-3p on RFA-deficient HCC cells. Metformin inhibited RFA-deficiency-induced HCC growth and tumor vascular abnormalities in vivo. Overall, metformin promotes the normalization of abnormal blood vessels after RFA deficiency in HCC by miR-302b-3p targeting TXNIP, which can be used to prevent the progression of HCC after RFA.

Metformin promotes the normalization of abnormal blood vessels after radiofrequency ablation deficiency in hepatocellular carcinoma by microRNA-302b-3p targeting thioredoxin-interacting protein.

Metformin has shown great promise in the treatment of HCC. Radiofrequency ablation (RFA) deficiency results in recurrence and metastasis of remaining HCC tumors. Here, we aimed to investigate the role and mechanism of metformin in HCC after RFA deficiency. HCC cell line Hep-G2 was selected to simulate RFA deficiency and named HepG2-H cells. After treating cells with different concentrations of metformin (2.5, 5, 10 μM) or transfecting related plasmids, cell proliferation, migration, invasion, apoptosis and angiogenesis were detected, in vitro permeability test was performed, and an angiogenesis-related protein VEGFA was analyzed. The residual HCC model after RFA deficiency was established in mice. Metformin was administered by gavage to detect changes in tumor volume and weight, and CD31 staining was used to observe microvessels. The targeting relationship between miR-302b-3p and TXNIP was demonstrated by the bioinformatics website, dual-luciferase reporter assay, and RNA pull-down assay. The results found that metformin inhibited RFA deficiency-induced growth and angiogenesis of HCC cells in vitro. miR-302b-3p counteracted the therapeutic effect of metformin on RFA deficiency. miR-302b-3p targeted regulation of TXNIP. The up-regulation of TXNIP reversed the effects of overexpression of miR-302b-3p on RFA-deficient HCC cells. Metformin inhibited RFA-deficiency-induced HCC growth and tumor vascular abnormalities in vivo. Overall, metformin promotes the normalization of abnormal blood vessels after RFA deficiency in HCC by miR-302b-3p targeting TXNIP, which can be used to prevent the progression of HCC after RFA.

Beneficial Effect of Metformin, Quercetin, and Resveratrol Combination on High Glucose-Induced lipogenesis in HepG2 Cells

Objectives: It has been reported that Metformin (MET), Resveratrol (RSV), and Quercetin (QRS) possess anti-lipogenic effects. This study aimed to investigate the combined effects of these compounds on lipid accumulation in HepG2 cells treated with high glucose (HG).
 Methods: HepG2 cells were treated with HG (33 mM), and different concentrations of MET, QRS, and RSV. The cytotoxic effects of these compounds were determined by an MTT assay. Changes in total lipid content and triglyceride (TG) levels were measured using Oil Red O staining and a triglyceride assay kit, respectively. The expression of fatty acid synthetase (FAS) and sterol regulatory element-binding protein 1c (SREBP1c) was evaluated by quantitative real-time PCR.
 Results: MET at doses 1, 2, and 5 mM, QRS at doses 5, 10, and 20 µM, and RSV at doses 25 and 50 µM could decrease total lipid content and triglyceride levels in HepG2 cells. The EC50 (half maximal effective concentration) from Oil red O staining results were MET: 1.786 mM, QRS: 8.132 μM, and RSV: 10.9 μM. The combination of MET (mM), QRS (µM), and RSV (µM) at ratios of (2:20:50), (1:10:25), and (0.5:5:10), could reduce lipogenesis greater than that observed with each of the individual compounds of MET, QRS, or RSV or the double combinations of MET+QRS or MET+RSV. In addition, combined treatment of MET (0.5mM), QRS (5 µM), and RSV (10 µM) was able to decrease SREBP-1c and FAS genes expression in HG-treated cells.
 Conclusion: The combination of MET, QRS, and RSV could inhibit lipid accumulation in HepG2 cells by reducing total lipid content, triglyceride levels, and the expression of the genes involved in lipogenesis.

Metformin attenuates inflammation and boosts autophagy in the liver and intestine of chronologically aged rats

BackgroundOur previous studies found that autophagy levels in liver and intestinal segments of naturally aging rats were downregulated, and the expression of pro-inflammatory factors was increased. The increased expression of pro-inflammatory factors might be related to the downregulation of autophagy. AMPK is the most critical upstream targeting and regulating molecule of autophagy, and Metformin, as an agonist of AMPK, has the effects of anti-inflammation and anti-aging. We pretreated 29-month-old naturally aging rats with Metformin for a short period and observed the changes in autophagy levels and pro-inflammatory factors in the liver, ileum, and colon after 31 days of intervention and preliminarily investigated the mechanism of its action. Methods29-month-old SPF male Wistar rats were divided into three groups: The control group, the Metformin 100 mg/kg intervention group, and the Metformin 250 mg/kg intervention group, with eight rats in each group. At 29 months, different concentrations of Metformin (100 mg/kg, 250 mg/kg) were given by gavage once a day until 30 months, and the control group was kept generally until 30 months. Western Blot was used to assess the expression levels of AMPK, P-AMPK, LC3, and P62 proteins in the liver and intestinal tissues. Intestinal and liver tissues were immunofluorescence labeled for LC3 and P62 proteins. Moreover, RT-qPCR was conducted to detect the expression levels of pro-inflammatory factors IL-1β, TNF-α, IL-6, and MMP-9 mRNA in liver and intestinal tissues. ResultsShort-term Metformin pretreatment (31 days) in naturally aging rats (29 months old) increased autophagy levels and down-regulated the expression of various pro-inflammatory cytokines (IL-1β, TNF-α, MMP-9, and IL-6) in various intestinal segments and the liver—the expression of LC3II protein enriched with the increase of Metformin concentration. The level of P62 protein decreased with the accumulation of Metformin concentration. And a higher concentration of Metformin was associated with increased expression of P-AMPK protein. ConclusionsMetformin intervention can boost the autophagy level in the liver and intestine and reduce the expression of aging-related inflammatory factors in aged rats, and these effects may be related to the increase of the AMPK phosphorylation level.

The enhancement of CRISPR/Cas9 gene editing using metformin

The CRISPR/Cas9 technology is a revolutionary tool that can be used to edit the genome. Specifically, the genome of hematopoietic stem cells (HSCs) could be edited to correct monogenic blood disorders as well as produce immunotherapies. However, the efficiency of editing HSCs remains low. To overcome this hurdle, we set out to investigate the use of metformin, an FDA-approved drug, to enhance gene modification. We assessed the effect of metformin on the growth of two hematopoietic cell lines: a myeloid-erythroid leukemic cell line (K562 cells) representative of the myeloid population and an immortalized T lymphocyte cell line (Jurkat cells) representative of the lymphoid population. No significant difference in growth patterns was observed in concentrations up to 10 mM metformin in both cell lines. We then assessed the ability of two different concentrations of metformin (0.001 mM or 1 mM), based on our observations, to enhance both (1) the cutting efficiency of Cas9 and (2) the targeting efficiency with the use of a donor DNA repair template. The cutting efficiency of Cas9 was significantly enhanced in a total of five guide RNAs (four specific to a platelet locus and one specific to an erythroid locus) following treatment. In addition, an enhancement in targeting was observed with the use of a GFP-containing donor DNA repair template with both concentrations. Overall, a greater than two-fold increase in GFP expression was noted in cells treated with metformin. This suggests that metformin, an FDA-approved drug, could be added to existing protocols to enhance CRISPR/Cas9 gene editing.

Metformin improves sheep sperm cryopreservation via vitalizing the AMPK pathway

Adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) is a key regulator of sperm function and physiological metabolism. Metformin, an inexpensive and effective antioxidant, is known to play an important role in the activation of AMPK. Therefore metformin has potential to improve sperm cryopreservation. The aim of this study was to investigate the effect of metformin during semen cryopreservation of sheep and to find the most effective concentration in freezing extender. Semen were cryopreserved with extender containing different concentrations of metformin (0, 0.25, 0.5, 1.0, 2.0 and 4.0 mmol/L). Sperm motility, acrosome integrity and plasma membrane integrity were measured after semen freezing and thawing. All results showed that sperm quality was significantly increased in the 1.0 mmol/L metformin-treated group compared with the control group (P < 0.05). In addition, the study showed that metformin effectively reduced the content of malondialdehyde (MDA) and reactive oxygen species (ROS), and increased the activity of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT) and total antioxidant capacity (T-AOC) of freeze-thawed sperm (P < 0.05). The optimal concentration of metformin was 1.0 mmol/L. Moreover, the results showed that AMPK was localized in the acrosome region, junction and midsection of sperm, and p-AMPK was distributed in the post-acrosomal region, junction and midsection. Western blot analysis indicated that 1.0 mmol/L metformin stimulated the phosphorylation of AMPK in sperm. Further results showed that 1.0 mmol/L metformin significantly increased the mitochondrial membrane potential (ΔΨm), ATP content, glucose uptake and lactate efflux of post-thawed sperm through the AMPK pathway, improved sperm quality, and increased the cleavage rate of in vitro fertilization (P < 0.05).

Effects of metformin on human gingival fibroblasts: an in vitro study

ObjectiveTo investigate the effects of metformin (MF) treatment on the matrix metalloproteinases (MMPs) and proinflammatory cytokines production from lipopolysaccharide (LPS) - stimulated human gingival fibroblasts (HGFs).MethodsHGFs were obtained from subcultures of biopsies from clinically healthy gingival tissues of patients undergoing oral surgeries. Cell cytotoxicity assay was used to determine the effect of different concentrations of MF on viability of HGFs. HGFs were then incubated and treated with different concentrations of MF and Porphyromonas gingivais (Pg) LPS. MMP-1, MMP-2, MMP-8, MMP-9, IL-1β, and IL-8 expression analysis was performed using xMAP technology (Luminex 200, Luminex, Austin, TX, USA). Student’s t-test for a single sample was used to compare the mean values of the study groups with the control value. A p-value of <0.05 and 95% confidence intervals were used to report the statistical significance and precision of mean values.ResultsConcentrations of 0.5, 1- and 2-mM MF had a minimal non-significant cytotoxic effect on the HGFs and caused statistically significant reduction of MMP-1, MMP-2, MMP-8 and IL-8 expressed by the LPS-stimulated HGFs.ConclusionThe results of the present study confirm that MF suppresses MMP-1, MMP-2, MMP-8 and IL-8 in LPS-stimulated HGFs suggesting an anti-inflammatory effect of MF and potential adjunct therapeutic role in the treatment of periodontal diseases.

Investigating the effects of Ceylon cinnamon water extract on HepG2 cells for Type 2 diabetes therapy.

Cinnamon and its extracts have been used as herbal remedies for many ailments, including for reducing insulin resistance and diabetes complications. Type 2 diabetes mellitus (T2DM) is a rapidly growing health concern around the world. Although many drugs are available for T2DM treatment, side effects and costs can be considerable, and there is increasing interest in natural products for managing chronic health conditions. Cinnamon may decrease the expression of genes associated with T2DM risk. The purpose of this study was to evaluate the effects of cinnamon water extract (CWE) compared with metformin on T2DM-related gene expression. HepG2 human hepatoma cells, widely used in drug metabolism and hepatotoxicity studies, were treated with different concentrations of metformin or CWE for 24 or 48 h. Cell viability was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay and glucose uptake was compared in untreated and CWE- or metformin-treated cells under high-glucose conditions. Finally, total RNA was extracted and analyzed by RNA sequencing (RNA-seq), and bioinformatics analyses were performed to compare the transcriptional effects of CWE and metformin. We found cell viability was better in cells treated with CWE than in metformin-treated cells, demonstrating that CWE was not toxic at tested doses. CWE significantly increased glucose uptake in HepG2 cells, to the same degree as metformin (1.4-fold). RNA-seq data revealed CWE and metformin both induced significantly increased (1.3- to 1.4-fold) glucose uptake gene expression compared with untreated controls. Transcriptional differences between CWE and metformin were not significant. The effects of 0.125 mg mL-1 CWE on gene expression were comparable to 1.5 mg mL-1 (9.5 mM) metformin. In addition, gene expression at 0.125 mg mL-1 CWE was comparable to 1.5 mg mL-1 (9.5 mM) metformin. Our results reveal that CWE's effects on cell viability, glucose uptake, and gene expression in HepG2 cells are comparable to those of metformin, suggesting CWE may be an effective dietary supplement for mitigating T2DM-related metabolic dysfunction.

Metformin enhances the osteogenic activity of rat bone marrow mesenchymal stem cells by inhibiting oxidative stress induced by diabetes mellitus: an in vitro and in vivo study.

The purpose of this study was to determine whether metformin (MF) could alleviate the expresssion of reactive oxygen species (ROS) and improve the osteogenic ability of bone marrow mesenchymal stem cells derived from diabetic rats (drBMSCs) in vitro, and to evaluate the effect of MF on the ectopic osteogenesis of drBMSCs in a nude mouse model in vivo. BMSCs were extracted from normal and diabetic rats. In vitro, a cell viability assay (Cell Counting Kit-8), tests of alkaline phosphatase (ALP) activity, and western blot analysis were first used to determine the cell proliferation and osteogenic differentiation of drBMSCs that were subjected to treatment with different concentrations of MF (0, 50, 100, 200, 500 μM). The cells were then divided into 5 groups: (1) normal rat BMSCs (the BMSCs derived from normal rats group), (2) the drBMSCs group, (3) the drBMSCs + Mito-TEMPO (10 μM, ROS scavenger) group, (4) the drBMSCs + MF (200 μM) group, and (5) the drBMSCs + MF (200 μM) + H2O2 (50 μM, ROS activator) group. Intracellular ROS detection, a senescence-associated β-galactosidase assay, ALP staining, alizarin red staining, western blotting, and immunofluorescence assays were performed to determine the effects of MF on oxidative stress and osteogenic differentiation in drBMSCs. In vivo, the effect of MF on the ectopic osteogenesis of drBMSCs was evaluated in a nude mouse model. MF effectively reduced ROS levels in drBMSCs. The cell proliferation, ALP activity, mineral deposition, and osteogenic-related protein expression of drBMSCs were demonstrably higher in the MF-treated group than in the non-MF-treated group. H2O2 inhibited the effects of MF. In addition, ectopic osteogenesis was significantly increased in drBMSCs treated with MF. MF promoted the proliferation and osteogenic differentiation of drBMSCs by inhibiting the oxidative stress induced by diabetes and enhenced the ectopic bone formation of drBMSCs in nude mice.

α-Hemihydrate calcium sulfate/n-hydroxyapatite combined with metformin promotes osteogenesis in vitro and in vivo.

This study aimed to examine the effects of loading different concentrations of metformin onto an α-hemihydrate calcium sulfate/nano-hydroxyapatite (α-CSH/nHA) composite. The material characteristics, biocompatibility, and bone formation were compared as functions of the metformin concentration. X-ray diffraction results indicated that the metformin loading had little influence on the phase composition of the composite. The hemolytic potential of the composite was found to be low, and a CCK-8 assay revealed only weak cytotoxicity. However, the metformin-loaded composite was found to enhance the osteogenic ability of MC3T3-E1 cells, as revealed by alkaline phosphate and alizarin red staining, real-time PCR, and western blotting, and the optimal amount was 500 µM. RNA sequencing results also showed that the composite material increased the expression of osteogenic-related genes. Cranial bone lacks muscle tissue, and the low blood supply leads to poor bone regeneration. As most mammalian cranial and maxillofacial bones are membranous and of similar embryonic origin, the rat cranial defect model has become an ideal animal model for in vivo experiments in bone tissue engineering. Thus, we introduced a rat cranial defect with a diameter of 5 mm as an experimental defect model. Micro-computed tomography, hematoxylin and eosin staining, Masson staining, and immunohistochemical staining were used to determine the effectiveness of the composite as a scaffold in a rat skull defect model. The composite material loaded with 500 µM of metformin had the strongest osteoinduction ability under these conditions. These results are promising for the development of new methods for repairing craniofacial bone defects.

Chlorogenic acid improves anti-lipogenic activity of metformin by positive regulating of AMPK signaling in HepG2 cells.

Metformin improves lipid profile, however, combination therapy is developing to increase its effectiveness and reduce the deleterious effects of metformin. Chlorogenic acid (CGA) has exhibited lipid-lowering effects. This study aimed to investigate the combined effect of metformin and CGA on lipid accumulation, as well as to elucidate the engaged mechanism in HepG2 cells. To find the non-lethal doses of metformin and CGA, MTT assay was performed. High Glucose (HG) at 33 mM was used to induce lipogenesis in HepG2 cells. Following treatment with different concentrations of metformin and CGA, total lipid content (Oil Red O-staining), triglyceride level, the genes expression of SREBP-1c and FAS, and phosphorylation of AMPK and ACC were measured. Both Metformin and CGA decreased HG-induced lipid accumulation individually, by decreasing total lipid content and triglyceride level. The lowest effective doses of metformin and CGA were 0.25 mM and 5 μM, respectively, which significantly reduced SREBP-1c and FAS genes expression. The combination of these concentrations reinforced these effects. The phosphorylation of AMPK and ACC were more increased by metformin in combination with CGA than both individually. Our findings suggest that CGA synergistically enhances metformin lipid reducing action via the regulating of involved factors in fatty acid synthesis. Therefore, co-administration of metformin with CGA may have further medical value in treating lipid metabolism disorders.

The Effect of Dual Bioactive Compounds Artemisinin and Metformin Co-loaded in PLGA-PEG Nano-particles on Breast Cancer Cell lines: Potential Apoptotic and Anti-proliferative Action.

The most prevalent malignancy among women is breast cancer. Phytochemicals and their derivatives are rapidly being recognized as possible cancer complementary therapies because they can modify signaling pathways that lead to cell cycle control or directly alter cell cycle regulatory molecules. The phytochemicals' poor bioavailability and short half-life make them unsuitable as anticancer drugs. Applying PLGA-PEG NPs improves their solubility and tolerance while also reducing drug adverse effects. According to the findings, combining anti-tumor phytochemicals can be more effective in regulating several signaling pathways linked to tumor cell development. The point of the study was to compare the anti-proliferative impacts of combined artemisinin and metformin on cell cycle arrest and expression of cyclin D1 and apoptotic genes (bcl-2, Bax, survivin, caspase-7, and caspase-3), and also hTERT genes in breast cancer cells. T-47D breast cancer cells were treated with different concentrations of metformin (MET) and artemisinin (ART) co-loaded in PLGA-PEG NPs and free form. The MTT test was applied to assess drug cytotoxicity in T47D cells. The cell cycle distribution was investigated using flow cytometry and the expression levels of cyclin D1, hTERT, Bax, bcl-2, caspase-3, and caspase-7, and survivin genes were then determined using real-time PCR. The findings of the MTT test and flow cytometry revealed that each state was cytotoxic to T47D cells in a time and dose-dependent pattern. Compared to various state of drugs (free and nano state, pure and combination state) Met-Art-PLGA/PEG NPs demonstrated the strongest anti-proliferative impact and considerably inhibited the development of T-47D cells; also, treatment with nano-formulated forms of Met-Art combination resulted in substantial downregulation of hTERT, Bcl-2, cyclin D1, survivin, and upregulation of caspase-3, caspase-7, and Bax, in the cells, as compared to the free forms, as indicated by real-time PCR findings. The findings suggested that combining an ART/MET-loaded PLGA-PEG NP-based therapy for breast cancer could significantly improve treatment effectiveness.

Metformin exerts an antitumor effect by inhibiting bladder cancer cell migration and growth, and promoting apoptosis through the PI3K/AKT/mTOR pathway

BackgroundTo observe and explore the effect of metformin on the migration and proliferation of bladder cancer T24 and 5637 cells in vitro.MethodsBladder cancer T24 and 5637 cell lines were cultured in vitro, and were divided into group A (blank control group) and group B (metformin group: 5, 10, 15, and 20 mmol/L); both groups were plated on 6-well plates at the same time. Culture in 24-well plates was used for wound healing assays and in 96-well plates for Transwell migration and invasion, and Cell Counting Kit-8 proliferation experiments. We observed and detected the cell migration and proliferation ability of each group at 48 h, and calculated the cell migration area and survival rate. Flow cytometry was used to detect cell apoptosis in the groups. The apoptosis-related proteins, cleaved-caspase 3, cleaved-PARP, and the PI3K/AKT/mTOR signaling pathway member proteins PI3K, phosphorylated (p)-PI3K, AKT, p-AKT, mTOR, and p-mTOR were detected using western blotting.ResultsAfter 48 h of treatment with different concentrations of metformin, the cell migration and proliferation capabilities were significantly lower than those in the blank control group. The proliferation and migration abilities of T24 and 5637 cells decreased in a metformin concentration-dependent manner (P < 0.05). The apoptosis rate under different concentrations of metformin, as detected by flow cytometry, showed a significantly higher rate in the metformin group than in the control group (P < 0.05). Compared with that in the control group, the level of cleaved-caspase 3 and cleaved-PARP protein in the metformin group was increased in each treatment group, and the levels of p-mTOR, p-AKT, and p-PI3K decreased significantly compared with those in the control group (P < 0.05).ConclusionMetformin inhibited bladder cancer T24 and 5637 cell migration and proliferation, and induced their apoptosis. The mechanism might involve inhibition of the activation of the PI3K/AKT/mTOR signaling pathway.

High-Concentration Metformin Reduces Oxidative Stress Injury and Inhibits the Growth and Migration of Clear Cell Renal Cell Carcinoma.

Objective To explore the mechanism of metformin in treating CCRCC. Methods Prospective cohort study was conducted. SOD and cyclin D in six CCRCC samples donated by volunteers were detected to compare the degree of oxidative stress injury and the status of cell proliferation. 786-0 CCRCC cells were cultured in vitro with different concentrations of metformin, and MTT assay and Transwell cell migration and wound healing assay were used to detect their proliferation and migration. After culture, SOD and cyclin D in 786-0 CCRCC cells were also detected. Results In the edge tissue, SOD was lower than in the tumor nest and normal tissue, and cyclin D was highly expressed. In grade II CCRCC, SOD was higher than in grade IV CCRCC, but cyclin D was also highly expressed in grade IV CCRCC. The cell proliferation rate and density of the metformin group were lower than the control group, while in the high-concentration metformin group, it was lower than medium- and low-concentration groups. After culture, the migration of 786-0 cells in the metformin group was significantly lower than that in the control group, the wound healing rate was decreased, and the migration and wound healing rates in the high-concentration metformin group were significantly lower than those in the medium- and low-concentration groups. However, the SOD of the metformin group was higher than the control group, but the cyclin D was lower, while the SOD was higher than medium- and low-concentration groups in the high-concentration group, but the cyclin D was lower after cultured. Conclusion High-concentration metformin can reduce oxidative stress injury, increase the expression of SOD in CCRCC, and reduce cyclin D in CCRCC to inhibit proliferation and migration, which has optimistic prospects and application value in controlling the progression of CCRCC.

[Metformin and lipopolysaccharide regulate transcription of NFATc2 gene via the transcription factor RUNX2].

To construct a luciferase reporter gene vector carrying human nuclear factor of activated T cells 2 (NFATc2) gene promoter and examine the effects of metformin and lipopolysaccharide (LPS) on the transcriptional activity of NFATc2 gene. The promoter sequence of human NFATc2 gene was acquired from UCSC website for PCR amplification. NFATc2 promoter fragment was inserted into pGL3-basic plasmid double cleaved with Kpn Ⅰ and Hind Ⅲ. The resultant recombinant plasmid pGL3-NFATC2-promoter was co-transfected with the internal reference plasmid pRL-TK in 293F cells, and luciferase activity in the cells was detected. Reporter gene vectors of human NFATc2 gene promoter with different fragment lengths were also constructed and assayed for luciferase activity. The changes in transcription activity of NFATc2 gene were assessed after treatment with different concentrations of metformin and LPS for 24 h. We also examined the effect of mutation in RUNX2-binding site in NFATC2 gene promoter on the regulatory effects of metformin and LPS on NFATc2 transcription. We successfully constructed pGL3-NFATc2-promoter plasmids carrying different lengths (2170 bp, 2077 bp, 1802 bp, 1651 bp, 1083 bp, 323 bp) of NFATc2 promoter sequences as verified by enzymatic digestion and sequencing. Transfection of 293F cells with the plasmid carrying a 1651 bp NFATc2 promoter (pGL3-1651 bp) resulted in the highest transcriptional activity of NFATc2 gene, and the luciferase activity was approximately 3.3 times that of pGL3-2170 bp (1.843 ± 0.146 vs 0.547 ± 0.085). Moderate (5 mmol/L) and high (10 mmol/L) concentrations of metformin significantly upregulated the transcriptional activity of pGL3-1651 bp by up to 2.5 and 3 folds, respectively. LPS at different doses also upregulated the transcriptional activity of pGL3-1651 bp by at least 1.6 folds. The mutation in the RUNX2 binding site on pGL3-1651 bp obviously reduced metformin- and LPS-induced enhancement of pGL3-1651bp transcription by 1.7 and 2 folds, respectively. pGL3-NFATc2-promoter can be transcribed and activated in 293F cells, and LPS and metformin can activate the transcription of pGL3- NFATc2-promoter in a RUNX2-dependent manner.

Metformin Eliminates CD133high/CD44high Prostate Cancer Stem Cells Via Cell Cycle Arrest and Apoptosis

Objectives: Cancer stem cells (CSCs), a small subpopulation of tumors, are responsible for chemo-radioresistance, metastasis, and cancer recurrence. The main aim of the present study is to investigate the potential effects of metformin on prostate CSCs (PCSCs). Methods: Flow cytometry was used to isolate cells with co-expression of CD133 and CD44. Sorted PCSCs were treated with different concentrations of metformin to determine the effects of metformin on cell viability using MTT assay. The association of cells exposure to metformin with apoptotic cell death and caspase activity, as well as cell cycle, were performed using the Muse Cell Analyzer. Results: In our study, for the first time we demonstrated the anti-cancer effects of metformin on PCSCs. Our results revealed that treatment with metformin reduced cell viability in CD133high/CD44high cells in a dose- and time-dependent manner. Metformin significantly induced early apoptosis and triggered the activity of several caspases associated with the apoptotic process. Metformin significantly altered the cell cycle distribution in CD133high/CD44high cells, leading to G0/G1 phase arrest. Conclusion: The results of the study revealed that metformin triggers cell death and apoptosis and modulates cell cycle distribution in CD133high/CD44high PCSCS. The present study raises the possibility that metformin is a potential anti-cancer agent for targeting PCSCs. Keywords: Prostate cancer, cancer stem cell, metformin

The effect of different concentrations of metformin in the liver and kidney functions for diabatic induced male rats.

The aimed of the study  to determination the effects of reducing diabetes drug "Metformin" on the liver and kidney function by number of biochemical parameters, including two liver enzymes: Alanine Transaminase (ALT), Alkaline phosphatase (ALP), and concentration of kidney function in the blood serum urea, and uric acid. male albino rats,which were used in this study. The results showed an increase in ALT activity, as well as the status of ALP activity. The concentrations of urea, and uric acid increase significantly (P≤0.05) in serum of rats the animals that were given alloxan compared to the control group. The concentrations of these enzymes decreased in the third and fourth groups of animals that were dosed with metformin at a concentration of 500,850 mg compared with the alloxan group. The group treated with metformin at a concentration of 850 mg was revealed three times the lower activities of all enzymes throughout the study period.

Metformin inhibits cholesterol‑induced adhesion molecule expression via activating the AMPK signaling pathway in vascular smooth muscle cells.

Recruitment of lymphocytes to the vascular wall contributes to the pathogenesis of atherosclerosis (AS). The expression of cellular adhesion molecules, such as vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, serves a critical role in mediating lymphocyte adhesion to the vascular wall. Cholesterol loading induces the expression of adhesion molecules in vascular smooth muscle cells (VSMCs), but the underlying mechanism is not completely understood. The present study aimed to investigate the mechanism underlying the effects of cholesterol on adhesion molecule expression, and whether metformin protected VSMCs against cholesterol-induced functional alterations. Human VSMCs were loaded with cholesterol and different concentrations of metformin. The expression levels of adhesion molecules were assessed via reverse transcription-quantitative PCR and western blotting. Reactive oxygen species (ROS) accumulation and levels were quantified via fluorescence assays and spectrophotometry, respectively. AMP-activated protein kinase (AMPK), p38 MAPK and NF-κB signaling pathway-related protein expression levels were evaluated via western blotting. Compared with the control group, cholesterol loading significantly upregulated adhesion molecule expression levels on VSMCs by increasing intracellular ROS levels and activating the p38 MAPK and NF-κB signaling pathways. Metformin decreased cholesterol-induced VSMC damage by activating the AMPK signaling pathway, and suppressing p38 MAPK and NF-κB signaling. The present study indicated the therapeutic potential of metformin for VSMC protection, reduction of monocyte adhesion, and ultimately, the prevention and treatment of AS.

Metformin pre-conditioning enhances the angiogenic ability of the secretome of dental pulp stem cells

The aim of the present study was to assess the influence of metformin on the angiogenic ability of secretomes from dental pulp stem cells. The stem cells were obtained from the dental pulp (DPSCs) (n = 3) using the explant culture method. We treated the DPSCs with different concentrations of metformin and assessed the expression of the angiogenesis-related genes. We also tested the angiogenic effect of the secretomes on the yolk sac membrane of the chick embryos by counting the quaternary blood vessel formations on the yolk sac membrane. We found that metformin treatment enhanced the angiogenic potential of the stem cell secretome in a dose-dependent manner. This was evidenced by the increase in the quaternary blood vessel formations in the yolk sac membrane with lower to higher concentrations of metformin. Pre-treatment with metformin modulates the angiogenic potential of the stem cell-conditioned media in a dose-dependent manner. The augmentation of the angiogenic potential of the DPSCs can aid regeneration, especially in scenarios requiring the regeneration of vacuoles.

Effects of different concentrations of metformin on osteoclast differentiation and apoptosis and its mechanism.

This study aimed to investigate the effect of metformin on osteoclast differentiation and apoptosis. Low concentration of metformin inhibited osteoclast differentiation and downregulated the expression of TRAP, RANK, Cathepsink, NFATC-1, MMP-9 and TRAF-6. High concentration of metformin promoted osteoclast apoptosis and upregulated the expression of Bax/Bcl-2 and caspase-3; BV/TV, BS/TV, Tb.N and BMD were increased while Tp.Sp decreased in the group of intraperitoneal metformin+femoral intramedullary osteoclast injection (Met+OC) compared with the control group, 1 nM metformin downregulated Akt, p44/42 MAPK, JNK, p38 MAPK phosphorylation, 5 nM metformin down regulated ERK and Akt phosphorylation. These results suggest that a low concentration of metformin inhibits osteoclast differentiation through PI3K/Akt and MAPK/ERK signaling pathway; high concentrations of metformin promote osteoclast apoptosis through PI3K/Akt and ERK signaling pathway.