Effective Inhibition of TNF-α/mTOR Axis-Mediated Liver Inflammation and Fibrosis Induced by TAA Using a Combination of Metformin and Resveratrol in Association with the Inhibition of the Profibrotic Gene and Protein Expression

SUMMARY: The response of the immune system to harmful stimuli leads to inflammation, and the adverse effects of the toxic hepatitis chemical, thioacetamide (TAA) on the human body are well documented. This article investigated the degree of protection provided by the combined pleotropic drug, metformin (Met) and the plant polyphenolic and the antiinflammatory compound, resveratrol (Res) on liver tissue exposed to TAA possibly via the inhibition of the inflammatory cytokine, tumor necrosis factor-α (TNF-α) / mammalian target of rapamycin (mTOR) axis-mediated liver fibrosis, as well as amelioration of profibrotic gene and protein expression. Rats were either given TAA (200 mg/kg via intraperitoneal injection) for 8 weeks beginning at the third week (experimental group) or received during the first two weeks of the experiment combined doses of metformin (200 mg/kg) and resveratrol (20 mg/kg) and continued receiving these agents and TAA until experiment completion at week 10 (treated group). A considerable damage to hepatic tissue in the experimental rats was observed as revealed by tissue collagen deposition in the portal area of the liver and a substantial increase (p<0.0001) in hepatic levels of the inflammatory marker, tumor necrosis factor-α (TNF-α), as well as blood levels of hepatocellular injury biomarkers, alanine aminotransferase (ALT) and aspartate aminotransferase (AST). TAA also augmented hepatic tissue levels of the signalling molecule that promotes liver fibrosis (mTOR), and profibrogenic markers; alpha-smooth muscle actin (α-SMA) protein, tissue inhibitor of metalloproteinases-1 (TIMP-1) mRNA, and matrix metalloproteinase-9 (MMP-9) mRNA. All these parameters were protected (p≤0.0016) by Met+Res. In addition, a significant correlation was detected between liver fibrosis score and inflammation, liver injury enzymes, mTOR, and profibrogenesis markers. Thus, these findings suggest that Met+Res effectively protect the liver against damage induced by thioacetamide in association with the downregulation of the TNF-α/mTOR/fibrosis axis.

La respuesta del sistema inmunológico a estímulos dañinos conduce a la inflamación y los efectos adversos de la tioacetamida (TAA), una sustancia química tóxica para el hígado, están bien documentadas. Este artículo investigó el grado de protección proporcionado por el fármaco pleotrópico combinado metformina (Met), el polifenólico vegetal y el compuesto antiinflamatorio resveratrol (Res) en el tejido hepático expuesto a TAA, posiblemente a través de la inhibición de la citoquina inflamatoria, factor de necrosis tumoral α (TNF-α)/objetivo de la fibrosis hepática mediada por el eje de rapamicina (mTOR), así como mejora de la expresión de genes y proteínas profibróticas. Las ratas recibieron TAA (200 mg/kg mediante inyección intraperitoneal) durante 8 semanas a partir de la tercera semana (grupo experimental) o recibieron durante las dos primeras semanas del experimento dosis combinadas de metformina (200 mg/kg) y resveratrol (20 mg/kg) y continuaron recibiendo estos agentes y TAA hasta completar el experimento en la semana 10 (grupo tratado). Se observó un daño considerable al tejido hepático en las ratas experimentales, como lo revela el depósito de colágeno tisular en el área portal del hígado y un aumento sustancial (p<0,0001) en los niveles hepáticos del marcador inflamatorio, el factor de necrosis tumoral-α (TNF- α), así como los niveles sanguíneos de biomarcadores de lesión hepatocelular, alanina aminotransferasa (ALT) y aspartato aminotransferasa (AST). TAA también aumentó los niveles en el tejido hepático de la molécula de señalización que promueve la fibrosis hepática (mTOR) y marcadores profibrogénicos; proteína actina del músculo liso alfa (α- SMA), inhibidor tisular de las metaloproteinasas-1 (TIMP-1) mRNA y matriz metaloproteinasa-9 (MMP-9) mRNA. Todos estos parámetros fueron protegidos (p≤0.0016) por Met+Res. Además, se detectó una correlación significativa entre la puntuación de fibrosis hepática y la inflamación, las enzimas de lesión hepática, mTOR y los marcadores de profibrogénesis. Por lo tanto, estos hallazgos sugieren que Met+Res protege eficazmente el hígado contra el daño inducido por la tioacetamida en asociación con la regulación negativa del eje TNF-α/mTOR/fibrosis.

Antitumor Effect of Metformin on Ovarian Cancer Cells / 肿瘤防治研究

Objective To explore the antitumor effects of metformin on ovarian cancer cells in vitro, particularly on tumor cell proliferation, cell cycle, apoptosis, migration, and possible mechanism. Methods Ovarian cancer cell lines (A2780, CAOV3, and SKOV3) were treated with different concentrations of metformin. Their proliferation was explored using the MTT and clone formation assays, cell migration was examined using the scratch and Transwell assays, and cell cycle and apoptosis were examined using flow cytometry. In addition, metformin’s effects on the phosphorylation of AMPK and mTOR and the expression of CXCR4 and Wnt/β-catenin protein was measured by Western blot. Results The survival rates of ovarian cancer cells decreased significantly with increasing metformin concentration and metformin treatment time. The IC50 values of metformin at 48 h for A2780, CAOV3, and SKOV3 cells were 16.36, 36.65, and 43.44 mmol/L, respectively. Compared with the control group, the clone formation ability and cell migration ability of ovarian cancer cells were significantly inhibited by metformin treatment and cell cycle arrested at the G0/G1 phase, and the apoptosis rate increased. As metformin concentration increased, the expression of phosphorylated AMPK protein gradually increased, and the expression levels of phosphorylated mTOR, CXCR4, Dvl3, β-catenin, cyclin D1, and CDK1 decreased. Conclusion Metformin exerts an antitumor effect on ovarian cancer cells, which is related to the activation of AMPK to inhibit CXCR4-mediated Wnt/β-catenin signaling pathway.

Tratamiento con metformina previene la adiposidad de la médula ósea femoral inducida por un síndrome metabólico experimental en ratas / Metformin treatment prevents experimental metabolic syndrome-induced femoral bone marrow adiposity in rats

RESUMEN Objetivo. Determinar el efecto de un tratamiento con metformina (MET) sobre la predisposición adipogénica de células progenitoras de médula ósea (CPMO), adiposidad de la médula ósea y propiedades biomecánicas óseas. Materiales y métodos. 20 ratas Wistar machos adultos jóvenes fueron separados en cuatro grupos, recibiendo en agua de bebida: 100% agua (C); 20% de fructosa (F); metformina 100 mg/kg peso/día (M); o fructosa más metformina (FM). Tras cinco semanas se sacrificaron los animales, se diseccionaron ambos húmeros para obtener CPMO, y ambos fémures para evaluar adiposidad medular (histomorfometría) y propiedades biomecánicas (flexión a 3 puntos). Las CPMO se cultivaron in vitro en medio adipogénico para evaluar expresión de RUNX2, PPAR-γ y RAGE por RT-PCR, actividad de lipasa y acumulación de triglicéridos. Resultados. La dieta rica en fructosa (grupo F) produjo un aumento tanto de triglicéridos in vitro, como de la adiposidad medular in vivo; siendo parcial o totalmente prevenido por un co-tratamiento con metformina (grupo FM). No se observaron diferencias en las pruebas biomecánicas femorales in vivo, ni en actividad de lipasa y relación RUNX2/PPAR-γ in vitro. La DRF aumentó la expresión de RAGE en CPMO, siendo prevenido por co-tratamiento con MET. Conclusiones. El síndrome metabólico inducido por una dieta rica en fructosa aumenta la adiposidad medular femoral y, en parte, la predisposición adipogénica de las CPMO. A su vez, esto puede ser prevenido total o parcialmente por un co-tratamiento oral con MET.

ABSTRACT Objective. To determine the effect of metformin (MET) treatment on adipogenic predisposition of bone marrow progenitor cells (BMPC), bone marrow adiposity and bone biomechanical properties. Materials and methods. 20 young adult male Wistar rats were sorted into four groups. Each of the groups received the following in drinking water: 100% water (C); 20% fructose (F); metformin 100 mg/kg wt/day (M); or fructose plus metformin (FM). After five weeks the animals were sacrificed. Both humeri were dissected to obtain BMPC, and both femurs were dissected to evaluate medullary adiposity (histomorphometry) and biomechanical properties (3-point bending). BMPC were cultured in vitro in adipogenic medium to evaluate RUNX2, PPAR-γ and RAGE expression by RT-PCR, lipase activity and triglyceride accumulation. Results. The fructose-rich diet (group F) caused an increase in both triglycerides in vitro, and medullary adiposity in vivo; being partially or totally prevented by co-treatment with metformin (group FM). No differences were found in femoral biomechanical tests in vivo, nor in lipase activity and RUNX2/PPAR-γ ratio in vitro. DRF increased RAGE expression in BMPC, being prevented by co-treatment with MET. Conclusions. Metabolic syndrome induced by a fructose-rich diet increases femoral medullary adiposity and, in part, the adipogenic predisposition of BMPC. In turn, this can be totally or partially prevented by oral co-treatment with MET.

Metformin Inhibits ROS/TNF-alpha Axis-Mediated Chronic Kidney Disease Induced by TAA Independent of Leukocyte Infiltration in Association with the Inhibition of Kidney Injury Biomarkers / La Metformina Inhibe la Enfermedad Renal Crónica Mediada por el Eje ROS/TNF-alfa Inducida por TAA Independientemente de la Infiltración de Leucocitos en Asociación con la Inhibición de Biomarcadores de Lesión Renal

SUMMARY: The toxic effects of thioacetamide (TAA) and carbon tetrachloride on the human body are well recognized. In this study, we examined whether TAA intoxication can induce kidney leukocyte infiltration (measured as leukocyte common antigen CD45) associated with the augmentation of the reactive oxygen species (ROS)/tumor necrosis factor-alpha (TNF-α) axis, as well as biomarkers of kidney injury with and without metformin treatment. Rats were either injected with TAA (200 mg/kg; twice a week for 8 weeks) before being sacrificed after 10 weeks (experimental group) or were pre-treated with metformin (200 mg/kg) daily for two weeks prior to TAA injections and continued receiving both agents until the end of the experiment, at week 10 (protective group). Using basic histology staining, immunohistochemistry methods, and blood chemistry analysis, we observed profound kidney tissue injury such as glomerular and tubular damage in the experimental group, which were substantially ameliorated by metformin. Metformin also significantly (p0.05) increase in kidney expression of CD45 positive immunostaining cells. In conclusion, we found that TAA induces kidney injury in association with the augmentation of ROS/TNF-α axis, independent of leukocyte infiltration, which is protected by metformin.

Son bien conocidosos los efectos tóxicos de la tioacetamida (TAA) y el tetracloruro de carbono en el cuerpo humano. En este estudio, examinamos si la intoxicación por TAA puede inducir la infiltración de leucocitos renales (medida como antígeno leucocitario común CD45) asociada con el aumento de las especies reactivas de oxígeno (ROS)/factor de necrosis tumoral-alfa (TNF-α), así como biomarcadores de daño renal con y sin tratamiento con metformina. A las ratas se les inyectó TAA (200 mg/kg; dos veces por semana durante 8 semanas) antes de sacrificarlas a las 10 semanas (grupo experimental) o se les pretrató con metformina (200 mg/kg) diariamente durante dos semanas antes de las inyecciones de TAA y continuaron recibiendo ambos agentes hasta el final del experimento, en la semana 10 (grupo protector). Usando tinción histológica básica, métodos de inmunohistoquímica y análisis químico de la sangre, observamos una lesión profunda del tejido renal, como daño glomerular y tubular en el grupo experimental, que mejoraron sustancialmente con la metformina. La metformina también inhibió significativamente (p0,05) en la expresión renal de células de inmunotinción positivas para CD45. En conclusión, encontramos que el TAA induce la lesión renal en asociación con el aumento del eje ROS/TNF-α, independientemente de la infiltración de leucocitos, que está protegida por metformina.

MALA: Acidosis láctica asociado al uso de metformina, una revisión a propósito de casos clínicos

La metformina es el fármaco preferido en el manejo inicial de la diabetes mellitus tipo 2 (DMT2). Aunque se recomienda su uso ampliamente, se debe tener precaución al prescribirla a poblaciones susceptibles a condiciones de riesgo de hipoperfusión sistémica, ya que puede provocar acumulación en el organismo y alteraciones metabólicas que desemboquen en acidosis láctica asociada a metformina, una complicación grave que a menudo es subdiagnosticada. Con el propósito de promover un mejor conocimiento sobre este tema, la presente revisión se centra en el análisis de la clínica, fisiopatología, diagnóstico y manejo de la acidosis láctica asociada a metformina, prestando especial atención al manejo mediante terapias de reemplazo renal. El análisis se basará en la experiencia de una serie de casos de acidosis láctica asociada a metformina atendidos en un centro clínico hospitalario en Chile.

Metformin is the preferred medication for the initial management of type 2 diabetes mellitus (T2DM). Although its use is widely recommended, caution should be exercised when prescribing it to populations susceptible to systemic hypoperfusion conditions, as it can lead to accumulation in the body and metabolic disturbances that may result in metformin-associated lactic acidosis. This severe complication is often underdiagnosed. To promote a better understanding of this topic, the present review focuses on the analysis of the clinical, pathophysiological, diagnostic, and management aspects of metformin-associated lactic acidosis, with particular attention to management through renal replacement therapies. The analysis will be based on the experience of a series of cases of metformin-associated lactic acidosis treated at a hospital clinical center in Chile.

Comparative evaluation of metformin & sitaformin in classic PCOS patients undergoing intracytoplasmic sperm injection: A randomized controlled pilot study

Background & objectives: Studies have shown that insulin resistance and hyperinsulinaemia play a major role in the pathogenesis of polycystic ovary syndrome (PCOS). Therefore, the use of insulin sensitizing drugs in the treatment of PCOS has attracted the attention of medicine and researchers. The aim of this study was to investigate the effects of sitaformin (sitagliptin/metformin) and metformin on the quality of oocyte and embryo in classic PCOS patients undergoing intracytoplasmic sperm injection (ICSI). Methods: Sixty patients of PCOS (25-35 yr) were randomly allocated into three groups (n=20, each group): a metformin-treated group (administered metformin 500 mg twice daily), a sitaformin-treated group (administered sitaformin 50/500 mg twice daily) and a placebo group. Participants in all the groups received the drug two months prior to the start of the ovulation cycle and treatment continued until the day of the oocyte aspiration. Results: Serum insulin and total testosterone levels decreaseed significantly after treatment in both the treatment groups as compared to the placebo (P<0.05). A significant decrease in the number of immature oocytes [MI + germinal vesicle (GV) stage] was observed in metformin and sitaformin groups as compared to the placebo. In addition, sitaformin group when compared to the metformin group showed a significant decrease in the number of immature oocytes (P<0.05). The number of mature and normal MII oocytes increased significantly in both the treatment groups compared to the placebo group (P<0.05). The number of mature and normal oocytes increased in sitaformin group in comparison to the metformin group, but the difference was not significant. There was a significant increase in the number of grade I embryos, fertilization and cleavage rates in the sitaformin group compared to the other groups (P<0.05). Interpretation & conclusions: This is the first study to compare the impact of sitaformin with metformin on oocyte and embryo quality in women with PCOS undergoing a gonadotropin-releasing hormone (GnRH) antagonist cycle. In conclusion, sitaformin can be more effective in decreasing immature oocytes and increasing the quality of embryos than the use of metformin.

Association of Metformin with Mortality in COVID-19 Patients: A Systematic Review and Meta-Analysis

Studies have demonstrated high prevalence of mortality in coronavirus disease (COVID-19) patients with type 2 diabetes mellitus; however, the effects of antidiabetic pharmacotherapy on COVID-19 complications need further exploration. The aim of the study was to explore the association of metformin use and mortality in COVID-19 patients. A literature search was conducted using the databases Medline (via PubMed) and Cochrane Central Register of Controlled Trials until February 09, 2021. Nine studies were included in the meta-analysis, including 12,684 COVID-19 patients. The meta-analysis suggested 37% lower risk of mortality in patients receiving metformin (risk ratio: 0.63; 95% confidence interval: 0.50–0.78; p?<?0.001). However, no significant difference in hospitalization days between the two groups (p?=?0.197) was observed. The analysis revealed significantly lower risk of having obesity (p?<?0.001), hypertension (p?<?0.001), heart failure (p?<?0.001), and cerebrovascular disease (p?=?0.015) in the group receiving metformin. The analysis also demonstrated significantly lower risk of using anticoagulants (p?=?0.015), diuretics (p?<?0.001), and antiplatelets (p?=?0.010) in patients receiving metformin. Our findings suggest that metformin use decreases mortality in COVID-19 patients. However, randomized studies demonstrating the consequences of metformin use are needed to understand the magnitude of the beneficial effects of metformin

Bioequivalence of metformin hydrochloride sustained-release tablets under fed condition in healthy subjects / 中国药科大学学报

@#To evaluate bioequivalence and safety of two kinds of metformin hydrochloride sustained-release tablets (test preparation vs reference preparation) under the condition of fed and single administration.A single center, randomized, open, single-dose, two-period, two-sequence, and double-crossover design was used.32 healthy subjects took 0.5 g of test preparation or reference preparation under fed and single-dose administration.4 mL of venous blood was collected from before administration (0 h) to 1, 3, 4, 4.5, 5, 5.5, 6, 7, 8, 9, 10, 12, 15, 24, 36 and 48 h after administration.The concentration of metformin in plasma samples was detected, and then the pharmacokinetic parameters were calculated by WinNonlin 7.0 software.When the 90% confidence intervals of cmax, AUC0-t and AUC0-∞ geometric mean ratio of test preparation and reference preparation were within 80.00%-125.00% equivalent intervals respectively, the bioequivalence of the two preparations was proved.One subject fell off due to adverse events.The main pharmacokinetic parameters of test preparation and reference preparation as follows: cmax were (0.68 ± 0.14) and (0.65 ± 0.11) mg/L, AUC0-t were (7.33 ± 1.65) and (7.00 ± 1.89) h·mg/L, AUC0-∞ were (7.39 ± 1.67) and (7.06 ± 1.91) h·mg/L, respectively.The 90% confidence intervals of the geometric mean ratio of the two main pharmacokinetic parameters were 101.45%-109.14%, 100.08%-112.32% and 100.24%-112.28%, respectively, which fell within the bioequivalence interval of 80.00%-125.00%.There were no serious adverse events and unexpected adverse events during the trial.The results show that test preparation and reference preparation are bioequivalent under fed and single-dose administration, safe and well tolerated in healthy subjects.

Hypoglycemic effect of electroacupuncture at "Tianshu" (ST 25) combined with metformin on rats with type 2 diabetes mellitus based on AMPK / 中国针灸

OBJECTIVE@#To observe the hypoglycemic effect of electroacupuncture (EA) at "Tianshu" (ST 25) combined with metformin on rats with type 2 diabetes mellitus (T2DM) as well as its effect on expression of adenosine monophosphate activated protein kinase (AMPK) in liver and pancreas.@*METHODS@#Thirty-six male SD rats were randomly divided into a blank group (6 rats) and a model establishing group (30 rats). The rats in the model establishing group were fed with high-fat diet and treated with intraperitoneal injection of low-dose streptozotocin (STZ) to establish T2DM model. The rats with successful model establishment were randomly divided into a model group, a control group, a metformin group, an EA group and a combination group, 6 rats in each group. The rats in the EA group were treated with EA at "Tianshu" (ST 25), dense-disperse wave, 2 Hz/15 Hz in frequency and 2 mA in current intensity, 20 min each time. The rats in the metformin group were treated with intragastric administration of metformin (190 mg/kg) dissolved in 0.9% sodium chloride solution (2 mL/kg). The rats in the combination group were treated with EA at "Tianshu" (ST 25) and intragastric administration of metformin. The rats in the control group were treated with intragastric administration of 0.9% sodium chloride solution with the same dose. All the treatments were given once a day for 5 weeks. After the intervention, the body mass and random blood glucose were detected; the serum insulin level was detected by ELISA; the expression of AMPK and phosphorylated adenosine monophosphate activated protein kinase (p-AMPK) in liver and pancreas was detected by Western blot method; the expression of protein gene product 9.5 (PGP9.5) was detected by immunofluorescence.@*RESULTS@#①Compared with the blank group, the body mass in the model group was decreased (P<0.05); compared with the model group, the body mass in the EA group and the combination group was decreased (P<0.05); the body mass in the EA group and the combination group was lower than the metformin group (P<0.05). Compared with the blank group, the random blood glucose in the model group was increased (P<0.01); compared with the model group, the random blood glucose in the metformin group, the EA group and the combination group was decreased (P<0.01). The random blood glucose in the combination group was lower than the metformin group and the EA group (P<0.05). ②Compared with the blank group, the insulin level in the model group was decreased (P<0.05); compared with the model group, the insulin level in the metformin group, the EA group and the combination group was all increased (P<0.05). The insulin level in the combination group was higher than the metformin group and the EA group (P<0.05). ③Compared with the blank group, the protein expression of AMPK and p-AMPK in liver tissue was decreased (P<0.05), and the protein expression of AMPK and p-AMPK in pancreatic tissue was increased (P<0.05) in the model group. Compared with the model group, the protein expression of AMPK and p-AMPK in liver tissue in the metformin group, the EA group and the combination group was increased (P<0.05, P<0.01); the protein expression of AMPK in pancreatic tissue in the metformin group was increased (P<0.05); the protein expression of AMPK in pancreatic tissue in the EA group and the combination group was decreased (P<0.05); the protein expression of p-AMPK in pancreatic tissue in the metformin group, the EA group and the combination group was decreased (P<0.05). The protein expression of AMPK and p-AMPK in liver tissue in the combination group was higher than that in the metformin group and the EA group (P<0.05); the protein expression of AMPK in pancreatic tissue in the EA group and the combination group was less than that in the metformin group (P<0.05), and the expression of p-AMPK protein in pancreatic tissue in the combination group was less than that in the metformin group and the EA group (P<0.05). ④Compared with the blank group, the expression of PGP9.5 in pancreatic tissue in the model group was increased (P<0.01); compared with the model group, the expression of PGP9.5 in pancreatic tissue in the metformin group, the EA group and the combination group was decreased (P<0.05, P<0.01). The expression of PGP9.5 in pancreatic tissue in the EA group was lower than the metformin group and the combination group (P<0.05).@*CONCLUSION@#Electroacupuncture at "Tianshu" (ST 25) could promote the effect of metformin on activating AMPK in liver tissue of T2DM rats, improve the negative effect of metformin on AMPK in pancreatic tissue, and enhance the hypoglycemic effect of metformin. The mechanism may be related to the inhibition of pancreatic intrinsic nervous system.

Metformin use and risk of ischemic stroke in patients with type 2 diabetes: A cohort study / 北京大学学报(医学版)

OBJECTIVE@#To explore the association between the use of metformin and the risk of ischemic stroke in patients with type 2 diabetes.@*METHODS@#A prospective cohort study was designed from the Fangshan family cohort in Beijing. According to metformin use at baseline, 2 625 patients with type 2 diabetes in Fangshan, Beijing were divided into metformin group or non-metformin group and the incidence of ischemic stroke between the different groups during follow-up was estimated and compared by Cox proportional hazard regression model. The participants with metformin were first compared with all the parti-cipants who did not use metformin, and then were further compared with those who did not use hypoglycemic agents and those who used other hypoglycemic agents.@*RESULTS@#The patients with type 2 diabetes were with an average age of (59.5±8.7) years, and 41.9% of them were male. The median follow-up time was 4.5 years. A total of 84 patients developed ischemic stroke during follow-up, with a crude incidence of 6.4 (95%CI: 5.0-7.7) per 1 000 person-years. Among all the participants, 1 149 (43.8%) took metformin, 1 476 (56.2%) were metformin non-users, including 593 (22.6%) used other hypoglycemic agents, and 883 (33.6%) did not use any hypoglycemic agents. Compared with metformin non-users, the Hazard ratio (HR) for ischemic stroke in metformin users was 0.58 (95%CI: 0.36-0.93; P = 0.024). Compared with other hypoglycemic agents, HR was 0.48 (95%CI: 0.28-0.84; P < 0.01); Compared with the group without hypoglycemic agents, HR was 0.65 (95%CI: 0.37-1.13; P=0.13). The association between metformin and ischemic stroke was statistically significant in the patients ≥ 60 years old compared with all the metformin non-users and those who used other hypoglycemic agents (HR: 0.48, 95%CI: 0.25-0.92; P < 0.05). Metformin use was associated with a lower incidence of ischemic stroke in the patients with good glycemic control (0.32, 95%CI: 0.13-0.77; P < 0.05). In the patients with poor glycemic control, and the association was not statistically significant (HR: 0.97, 95%CI: 0.53-1.79; P>0.05). There was an interaction between glycemic control and metformin use on incidence of ischemic stroke (Pinteraction < 0.05). The results of the sensitivity analysis were consistent with the results in the main analysis.@*CONCLUSION@#Among patients with type 2 diabetic in rural areas of northern China, metformin use was associated with lower incidence of ischemic stroke, especially in patients older than 60 years. There was an interaction between glycemic control and metformin use in the incidence of ischemic stroke.

The development and benefits of metformin in various diseases / 医学前沿

Metformin has been used for the treatment of type II diabetes mellitus for decades due to its safety, low cost, and outstanding hypoglycemic effect clinically. The mechanisms underlying these benefits are complex and still not fully understood. Inhibition of mitochondrial respiratory-chain complex I is the most described downstream mechanism of metformin, leading to reduced ATP production and activation of AMP-activated protein kinase (AMPK). Meanwhile, many novel targets of metformin have been gradually discovered. In recent years, multiple pre-clinical and clinical studies are committed to extend the indications of metformin in addition to diabetes. Herein, we summarized the benefits of metformin in four types of diseases, including metabolic associated diseases, cancer, aging and age-related diseases, neurological disorders. We comprehensively discussed the pharmacokinetic properties and the mechanisms of action, treatment strategies, the clinical application, the potential risk of metformin in various diseases. This review provides a brief summary of the benefits and concerns of metformin, aiming to interest scientists to consider and explore the common and specific mechanisms and guiding for the further research. Although there have been countless studies of metformin, longitudinal research in each field is still much warranted.

Leaky Gut Plays a Critical Role in the Pathophysiology of Autism in Mice by Activating the Lipopolysaccharide-Mediated Toll-Like Receptor 4-Myeloid Differentiation Factor 88-Nuclear Factor Kappa B Signaling Pathway / 神经科学通报·英文版

Increased intestinal barrier permeability, leaky gut, has been reported in patients with autism. However, its contribution to the development of autism has not been determined. We selected dextran sulfate sodium (DSS) to disrupt and metformin to repair the intestinal barrier in BTBR T+tf/J autistic mice to test this hypothesis. DSS treatment resulted in a decreased affinity for social proximity; however, autistic behaviors in mice were improved after the administration of metformin. We found an increased affinity for social proximity/social memory and decreased repetitive and anxiety-related behaviors. The concentration of lipopolysaccharides in blood decreased after the administration of metformin. The expression levels of the key molecules in the toll-like receptor 4 (TLR4)-myeloid differentiation factor 88 (MyD88)-nuclear factor kappa B (NF-κB) pathway and their downstream inflammatory cytokines in the cerebral cortex were both repressed. Thus, "leaky gut" could be a trigger for the development of autism via activation of the lipopolysaccharide-mediated TLR4-MyD88-NF-κB pathway.

Effects of plateau hypoxia on population pharmacokinetics and pharmacodynamics of metformin in patients with Type 2 diabetes / 中南大学学报(医学版)

OBJECTIVES@#Metformin is the basic drug for treating diabetes, and the plateau hypoxic environment is an important factor affecting the pharmacokinetics of metformin, but there have been no reports of metformin pharmacokinetic parameters in patients with diabetes mellitus type 2 (T2DM) in the high-altitude hypoxic environment. This study aims to investigate the effect of the hypoxic environment on the pharmacokinetics and assess the efficacy and safety of metformin administration in patients with Type 2 diabetes mellitus (T2DM).@*METHODS@#A total of 85 patients with T2DM taking metformin tablets in the plateau group (n=32, altitude: 1 500 m) and control group (n=53, altitude: 3 800 m) were enrolled according to the inclusion and exclusion criteria, and 172 blood samples were collected in the plateau group and the control Group. A ultra-performance liquid chromatography/tandem mass spectrometry (UFLC-MS/MS) method was established to determine the blood concentration of metformin, and Phoenix NLME software was used to establish a model of pharmacokinetics of metformin in the Chinese T2DM population. The efficacy and serious adverse effects of metformin were compared between the 2 groups.@*RESULTS@#The population pharmacokinetic modeling results showed that plateau hypoxia and age were the main covariates for model building, and the pharmacokinetic parameters were significantly different between the plateau and control groups (all P<0.05), including distribution volume (V), clearance (CL), elimination rate constant (Ke), half-life(T1/2), area under the curve (AUC), time to reach maximum concentration (Tmax). Compared with the control group, AUC was increased by 23.5%, Tmax and T1/2 were prolonged by 35.8% and 11.7%, respectively, and CL was decreased by 31.9% in the plateau group. The pharmacodynamic results showed that the hypoglycaemic effect of T2DM patients in the plateau group was similar to that in the control group, the concentration of lactic acid was higher in the plateau group than that in the control group, and the risk of lactic acidosis was increased after taking metformin in the plateau population.@*CONCLUSIONS@#Metformin metabolism is slowed down in T2DM patients in the hypoxic environment of the plateau; the glucose-lowering effect of the plateau is similar, and the attainment rate is low, the possibility of having serious adverse effects of lactic acidosis is higher in T2DM patients on the plateau than on the control one. It is probably suggested that patients with T2DM on the plateau can achieve glucose lowering effect by extending the interval between medication doses and enhancing medication education to improve patient compliance.

Metformin HCl Oral Preparation Exhibits Anticancer Activity In-vitro in a Human Non-small Cell Lung Tumour Cell Line

@#Introduction: The most common variety of lung cancer is non–small cell lung cancer (NSCLC) accounting for 84% of new cases. Surgery, chemotherapy and radiation are the primary treatment option. Metformin has recently been demonstrated to have an anti-tumour impact on various cancer cells. The goal of this investigation was to determine the growth inhibitory, antiproliferative, cytotoxic, apoptotic and cell cycle arrest properties of metformin HCl oral tablets on the A549 lung carcinoma cell line. Methods: The cells were treated with different dosages of an oral preparation of metformin, with untreated cells used as a control. The Trypan Blue Exclusion Assay was used to determine metformin’s inhibitory and cytotoxic effects. Flow cytometry was used to evaluate apoptosis and cell cycle arrest. Results: In a dose-dependent manner, metformin HCl was able to reduce the viability of treated cells compared to the untreated control. Cell proliferation was considerably inhibited in the treated group with the IC50 dose than in the untreated control group and the IC50 dose showed no cytotoxic effect on L929 cells. Induction of apoptosis and cell cycle arrest was observed in the IC50 dose-treated group by Flow cytometry analysis and data showed metformin oral drug causes early apoptosis and a considerable cell increase in the S phase of the cell cycle. Conclusion: Metformin inhibits cell growth and induces apoptosis and cell cycle arrest in the cell line. A comprehensive proteome examination is required to understand more about the mechanism of action of the oral metformin HCl on cancer cells

Progress of mechanism of metformin against colorectal cancer / 肿瘤研究与临床

Colorectal cancer is a common gastrointestinal malignant tumor with morbidity and mortality rising year by year. In recent years, the studies in and out of China have reported that metformin could inhibit the growth of colorectal cancer cells and improve the prognosis of patients by indirectly reducing the levels of insulin and glucose in the blood, or directly activating the AMP-activated protein kinase signaling pathways, promoting apoptosis of tumor cells, enhancing sensitivity to chemotherapy, inhibiting inflammatory responses, affecting the intestinal flora, and regulating the immune function, etc. This article reviews the current research status and controversies related to metformin against colorectal cancer, in an effort to provide new evidences for the treatment of colorectal cancer.

Metformin inhibiting the activation of NLRP3 inflammasome and pyroptosis in diabetic retinal vascular endothelial cells / 中华眼底病杂志

Objective:To observe the effect of metformin (Met) on inflammatory bodies and focal death in human retinal microvascular endothelial cells (hRMEC) in diabetes mellitus (DM) microenvironment.Methods:Experimental research was divided into in vivo animal experiment and in vitro cell experiment. In vivo animal experiments: 9 healthy C57BL/6J male mice were randomly divided into DM group, normal control group, and DM+Met group, with 3 mice in each group. DM group and DM+Met group mice were induced by streptozotocin to establish DM model, and DM+Met group was given Met 400 mg/ (kg · d) intervention. Eight weeks after modeling, the expression of NLRP3, cleaved-membrane perforating protein D (GSDMD) and cleaved-Caspase-1 in the retina of mice in the normal control group, DM group and DM+Met group were observed by immunohistochemical staining. In vitro cell experiments: hRMEC was divided into conventional culture cell group (N group), advanced glycation end products (AGE) group, and AGE+Met group. Joining the AGE, AGE+Met groups cells were induced by 150 μg/ml of glycation end products, and 2.0 mmol/L Met was added to the AGE+Met group. Pyroptosis was detected by flow cytometry; 2' ,7'-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe was used to detect the expression of reactive oxygen species (ROS) in cells of each group. Real-time fluorescence quantitative polymerase chain reaction and Western blot were used to detect the relative mRNA and protein expression levels of NLRP3, cleaved-GSDMD, cleaved-Caspase-1 in each group of cells. Single factor analysis of variance was used for comparison among the three groups.Results:In vivo animal experiments: compared with the DM group, the expression of NLRP3, cleaved-GSDMD, and cleaved-Caspase-1 in the retina of normal control group and DM+Met group mice was significantly reduced, with significant difference among the 3 groups ( F=43.478, 36.643, 24.464; P<0.01). In vitro cell experiment and flow cytometry showed that the pyroptosis rate of AGE group was significantly higher than that of N group and AGE+Met group ( F=32.598, P<0.01). The DCFH-DA detection results showed that the intracellular ROS levels in the N group and AGE+Met group were significantly lower than those in the AGE group, with the significant difference ( F=47.267, P<0.01). The mRNA ( F=51.563, 32.192, 44.473; P<0.01) and protein levels ( F=63.372, 54.463, 48.412; P<0.01) of NLRP3, cleaved-GSDMD, and cleaved-Caspase-1 in hRMEC of the AGE+Met group were significantly reduced compared to the N group. Conclusion:Met can down regulate the expression of NLRP3 inflammatory body related factors in hRMEC and inhibit pyroptosis.

Effect of metformin on the polarization status of microglia and photoreceptor cells activity in a high glucose environment / 中华眼底病杂志

Objective:To observe the effect of metformin on the polarization state and photoreceptor cell activity of microglia (BV2 cells) in a high glucose environment.Methods:An experimental study. BV2 cells were divided into a control group, a high glucose group, and a metformin+high glucose group. The cells in the high glucose group were cultured with 75 mmol/L glucose in the medium; the cells in the metformin+high glucose group were pretreated with 2 mmol/L metformin for 12 h and then placed in 75 mmo/L glucose concentration medium. The relative expression of M1 marker inducible nitric oxide synthase (iNOS), CD86 and M2 markers arginase 1 (Arg-1), and CD206 protein were detected by Western blot. Interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-4 were detected by enzyme-linked immunosorbent assay (ELISA). BV2 cells were co-cultured with mouse retinal photoreceptor cells (661W cells) for 24 h. The proliferation rate of 661W cells in each group was measured by methyl thiazolyl tetrazolium (MTT) colorimetric assay; the apoptosis rate of 661W cells in each group was measured by flow cytometry and terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL). An independent sample t-test was used for comparison between groups. Results:Western blot assay showed that the relative expression of iNOS and CD86 protein was increased and the relative expression of Arg-1 and CD206 protein was decreased in BV2 cells in the high glucose group compared with the control group, and the differences were all statistically significant ( t=-16.783, -11.605, 4.325, 4.649; P<0.05); compared with the high glucose group, the relative expression of iNOS and CD86 protein was decreased and the relative expression of Arg-1 and CD206 protein was increased in BV2 cells in the metformin + high glucose group compared with the high glucose group, and the differences were all statistically significant ( t=7.231, 5.560, -8.035, -8.824; P<0.01). ELISA results showed that compared with the control group, the BV2 cells in the high glucose group had increased IL-6, TNF-α content and IL-4 content was decreased in BV2 cells in the high glucose group compared with the control group, and the differences were all statistically significant ( t=-64.312, -127.147, 71.547; P<0.001); compared with the high glucose group, IL-6 and TNF-α content was significantly decreased and IL-4 content was significantly increased in BV2 cells in the metformin+high glucose group, and the differences were all statistically significant ( t=44.426, 83.232,-143.115; P<0.001). After co-culture of BV2 cells with 661W cells for 24 h, the results of MTT colorimetric assay showed that compared with the control group, the activity of 661W cells in the high glucose group was significantly reduced, and the difference was statistically significant ( t=7.456, P<0.01); compared with the high glucose group, the activity of 661W cells in the metformin+high glucose group was increased ( t=-3.076, P<0.05). TUNEL method and flow cytometry showed that the apoptosis rate of 661W cells in the high glucose group was significantly higher compared with the control group, and the differences were both statistically significant ( t=-22.248, -22.628; P<0.001); compared with the high glucose group, the apoptosis rate of 661W cells in the metformin+high glucose group was significantly decreased, and the difference was statistically significant ( t=11.767, 6.906; P<0.001, 0.01). Conclusion:In the high glucose environment, metformin inhibited the inflammatory response and attenuated the apoptosis of photoreceptor cells by regulating the polarization of microglia toward the M2 type.

Effect of metformin preconditioning on AMPK/PINK1 signaling pathway during myocardial ischemia-reperfusion injury in diabetic rats / 中华麻醉学杂志

Objective:To evaluate the effect of metformin preconditioning on adenosine monophosphate-activated protein kinase(AMPK)/PTEN-induced putative protein kinase(PINK1) signaling pathway during ischemia-reperfusion (I/R) injury in diabetic rats.Methods:Thirty-six clean-grade healthy male Sprague-Dawley rats, aged 6 weeks, weighing 120-160 g, were divided into 3 groups ( n=12 each) by the random number table method: diabetic sham operation group (DS group), diabetic myocardial I/R group (DI/R group) and diabetic myocardial I/R+ metformin preconditioning group(DI/R+ Met group). After 4 weeks of feeding a high-fat and high-glucose diet, the model of type 2 diabetes mellitus was induced by a single intraperitoneal injection of 1% streptozotocin 40 mg/kg. The myocardial I/R injury was induced by blocking the anterior descending branch of the left coronary artery for 30 min followed by 120-min reperfusion in anesthetized animals. In DI/R+ Met group, metformin 200 mg/kg was given by intragastric gavage once a day within 1 week before myocardial ischemia. Blood samples from the femoral vein were collected at 120 min of reperfusion for determination of the serum creatine kinase isoenzymes (CK-MB) and cardiac troponin I (cTnI) concentrations by enzyme-linked immunosorbent assay. Then the rats were sacrificed and myocardial tissues were obtained for examination of the pathological changes(by HE staining) and for determination of the percentage of myocardial infarct size (by the double staining of Ewan blue and TTC) and expression of myocardial autophagy-related protein Beclin-1, PTEN-induced putative kinase 1 (PINK1), phosphorylated 5′-adenosine monophosphate-activating protein kinase (p-AMPK), and ratio of microtubule-associated protein 1 light chain 3Ⅱ/Ⅰ (LC3Ⅱ/Ⅰ) (by Western blot). Results:Compared with DS group, the percentage of myocardial infarct size and serum CK-MB and cTnI concentrations were significantly increased, the expression of Beclin-1, p-AMPK and PINK1 in myocardial tissues was up-regulated, the ratio of LC3II/I was increased( P<0.05), and the pathological changes were aggravated in DI/R group and DI/R+ Met group. Compared with DI/R group, the percentage of myocardial infarct size and serum CK-MB and cTnI concentrations were significantly decreased, the expression of Beclin-1, p-AMPK and PINK1 in myocardial tissues was up-regulated, the ratio of LC3Ⅱ/Ⅰ was increased ( P<0.05), and the pathological changes were significantly reduced in DI/R+ Met group. Conclusions:The mechanism by which metformin preconditioning reduces myocardial I/R injury is related to activation of AMPK/PINK1 signaling pathway and up-regulation of mitochondrial autophagy in diabetic rats.

Roles and mechanisms of metformin in improving cognitive dysfunction induced by chronic cerebral hypoperfusion in rats / 中华行为医学与脑科学杂志

Objective:To explore the roles and mechanisms of metformin in the improvement of cognitive dysfunction induced by chronic cerebral hypoperfusion in rats.Methods:Total 82 SD male rats (SPF grade) aged 3-4 months were randomly divided into four groups: sham operation control group (Con group, n=15), sham operation with metformin treatment group (Met group, n=20), 2-vessel occlusion group (2VO group, n=22), and 2-vessel occlusion with metformin administration group (2VO+ Met group, n=25). The chronic cerebral hypoperfusion model was established by bilateral common carotid artery ligation, and the carotid arteries of rats in Con group and Met group were only separated without ligation.After 2VO operation, rats in 2VO+ Met group and Met group were given metformin solution in drinking water at a dose of 100 mg/kg per day for 4 weeks.After 4-week continuous intervention with metformin, Morris water maze was performed to test the spatial cognitive function of the rats, in vivo electrophysiological technology was used to detect the long-term potential of the rats, and enzyme-linked immunosorbent assay(ELISA) was used to detect the concentrations of inflammatory factor tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β) and interleukin-6(IL-6) in the hippocampus.The density of dendritic spines of hippocampal neurons was observed by Golgi staining, and the synaptic structure of hippocampal neurons, especially the vesicle density, was observed by transmission electron microscopy.SPSS 16.0 software was used for statistical analysis.Repetitive measurement ANOVA was used for the escape latency data of 7 days repeated learning training in water maze.One-way ANOVA was used for the comparison of other data among multiple groups, and Dunnett's t test was used for further pairwise comparison. Results:Morris water maze results showed that during 7 days of learning training, the time and group interaction for escape latency was not significant in the 4 groups of rats ( F=0.93, P>0.05), but the time main effect ( F=25.90, P<0.05) and group main effect ( F=13.20, P<0.05) were significant.Morris water maze test showed that from the 3rd to 7th day, the escape latencies in 2VO group were significantly longer than those in Con group and 2VO+ Met group(all P<0.05). The short-term memory of rats was detected after 1 day of rest.The results showed that the escape latency in 2VO group was significantly longer than that in Con group and 2VO + Met group( P<0.01). The retention time and crossing times in the platform area of 2VO rats were less than those in Con group and 2VO + Met group ( P<0.01). Electrophysiological results showed that the relative field excitatory postsynaptic potential slope of 2VO group (1.29±0.09) was significantly lower than that in Con group (2.07±0.09) and 2VO + Met group (1.69±0.08)( P<0.01). ELISA results showed that TNF-α level in hippocampal tissue of 2VO group was significantly higher than that in Con group and 2VO+ Met group; IL-1β and IL-6 levels in hippocampal tissue of 2VO group were significantly higher than those in Con group and 2VO + Met group.Density of dendritic spines in hippocampal neurons of 2VO group was significantly lower than that in Con group and 2VO+ Met group.The density and proportion of immature dendritic spines in hippocampal neurons of 2VO group were significantly higher than those in Con group and 2VO + Met group.Synaptic vesicle density of neurons in CA1 area of hippocampus in 2VO group ((230.29±19.44) vescicles/μm 2) was significantly lower than that in the Con group ((414.52±13.17) vescicles/μm 2) and 2VO+ Met group ((313.19±12.42) vescicles/μm 2). Conclusion:Metformin can reduce neuroinflammation of hippocampus with chronic cerebral hypoperfusion and improve synaptic plasticity and cognitive dysfunction.It may have potential application value in the treatment of vascular cognitive dysfunction.

Clinical study on dapagliflozin combined with metformin for the treatment of type 2 diabetes mellitus / 中国基层医药

Objective:To investigate the clinical effect of dapagliflozin combined with metformin on type 2 diabetes mellitus (T2DM).Methods:A total of 100 patients with T2DM who received treatment in The Second People's Hospital of Hefei from June 2019 to May 2021 were included in this study. They were randomly divided into a control group ( n = 50) and an experimental group ( n = 50). The control group was treated with metformin, and the experimental group was treated with dagglitazin combined with metformin. All patients were treated for 3 months. Blood glucose index, blood lipid level, and the incidence of adverse reactions were compared between the two groups. Results:After treatment, fasting blood glucose, 2-hour post-prandial blood glucose, and glycosylated hemoglobin in the experimental group were (5.56 ± 0.37) mmol/L, (8.32 ± 0.23) mmol/L, and (6.17 ± 0.26)% respectively, which were significantly lower than (6.96 ± 0.48) mmol/L, (9.58 ± 0.39) mmol/L, and (7.27 ± 0.26)% respectively in the control group ( t = 3.59, 6.92, 5.03, all P < 0.05). The total cholesterol and triglyceride in the experimental group were (3.58 ± 0.53) mmol/L and (1.25±0.26) mmol/L, respectively, which were significantly lower than (4.94 ± 0.58) mmol/L and (1.93 ± 0.18) mmol/L in the control group ( t = 3.16, 4.25, both P < 0.05). There was no significant difference in the incidence of adverse reactions between the two groups ( P > 0.05). Conclusion:Dapagliflozin combined with metformin can effectively control blood glucose and blood lipid in T2DM patients without increasing adverse reactions.