Atherosclerotic lesions contain heterogeneous macrophage populations with divergent functional phenotypes that play opposing roles in disease progression, creating a major obstacle for the development of macrophage-targeted therapy. The identification of novel molecules that exhibit both preferential expression and functional activity in inflammatory plaque macrophages is crucial for the development of more specific anti-inflammatory therapies against atherosclerosis. Here, we report that the expression of C-C motif chemokine receptor-like 2 (CCRL2), a nonsignaling atypical chemokine receptor initially cloned from LPS-activated macrophages, progressively increased in atherosclerotic lesions during disease progression in both human and mouse models, with predominant expression in inflammatory macrophages characterized by high expression of NLRP3 inflammasome components. Moreover, specific deletion of hematopoietic CCRL2 attenuated atherosclerotic progression in Apoe-/- mice fed a high-cholesterol diet, as evidenced by significant decreases in plaque burden, macrophage accumulation, and NLRP3 inflammasome activation within the plaques, without affecting blood lipid levels. Mechanistically, ox-LDL or other danger signals induce macrophages to express CCRL2, which can be endocytosed and relocalized to early endosomes, where it interacts with NLRP3 to promote inflammasome assembly and activation. Importantly, macrophage-targeted nanoparticle delivery of Ccrl2-siRNA effectively attenuated atherosclerotic progression in vivo, concomitant with reduced levels of IL-1β, the key effector cytokine of the NLRP3 inflammasome, and diminished macrophage accumulation within plaques. Taken together, the results of our study establish CCRL2 as both a new marker for identifying inflammatory macrophages and a promising anti-inflammatory therapeutic target for atherosclerosis through the modulation of aberrant NLRP3 inflammasome activation in pathogenic inflammatory macrophages.

Background Finerenone is a third-generation mineralocorticoid receptor antagonists, which has shown good cardiac function improvement in patients with type 2 diabetes in large-scale clinical trials. However, its specific role in diabetic cardiomyopathy remains unclear. We explored the potential functions and mechanisms of finerenone in diabetic cardiomyopathy. Methods The type 2 diabetic rat model was induced by high-fat diet and low-dose streptozotocin (n = 6, each group). Next the drug group was treated with finerenone (1 mg/kg/day) for 8 weeks. Then we detected the cardiac structure and function and relevant indicators. Neonatal rat cardiomyocytes were used for in vitro culture to determine the direct effect of finerenone on cardiomyocytes stimulated by high glucose and high fatty acid. Results Compared with the control group, rats in the type 2 diabetes group exhibited hyperglycemia, hyperlipidemia, and impaired cardiac function. Myocardium showed increased fibrosis and apoptosis. Finerenone attenuated these impairments without changing blood glucose levels. In neonatal rat cardiomyocytes, the stimulation of high concentrations of palmitic acid increased fatty acid uptake, as well as increased reactive oxygen species and apoptosis. Finerenone significantly improved fatty acid metabolism, reduced cellular inflammation levels, and decreased apoptosis. Conclusions By blocking the mineralocorticoid receptor, finerenone attenuates cardiac steatosis, myocardial fibrosis and apoptosis, and subsequent myocardial remodeling and diastolic dysfunction in type II diabetic rats.

Background It is widely accepted that omega-3 fatty acids are beneficial in the prevention of cardiovascular disease, but many large randomized controlled trial studies and meta-analyses have come to different conclusions. The evidence for omega-3 fatty acids supplementation to prevent cardiovascular disease remains insufficient. We conducted a systematic review and meta-analysis to evaluate the efficacy and safety of several types of omega-3 fatty acids supplements. Methods We comprehensively searched the online database and found 15 RCTs. The primary efficacy outcomes included major cardiovascular events, myocardial infarction, heart failure, atrial fibrillation, stroke, cardiovascular death, and all-cause death. The safety endpoints included gastrointestinal problems, bleeding-related disorders, and cancer. Subgroup analysis was conducted according to the main characteristics of the population, and the dose-response relationship of omega-3 fatty acids was evaluated by meta-regression. All results were calculated by the random effect model. Statistical heterogeneity was assessed using chi-square tests and quantified using I-square statistics. Results The incidence of major cardiovascular events (RR 0.95, 95%CI 0.91 to 0.99, P = 0.026), myocardial infarction (RR 0.90, 95%CI 0.83 to 0.98; P = 0.021), and cardiovascular death (RR 0.94, 95%CI 0.88 to 0.99; P = 0.028) was reduced in the omega-3 fatty acid group compared with the control group. An increased risk of atrial fibrillation (RR 1.25, 95%CI 1.10 to 1.41; P = 0.000) was observed in patients in the omega-3 fatty acid group. No statistical differences were observed between the two groups in heart failure, stroke, and all-cause death. For safety endpoints, there were no statistically significant differences between the two groups in gastrointestinal problems, bleeding-related disorders, and cancer. Subgroup analysis showed that the cardiovascular benefit of omega-3 fatty acids was primarily attributable to the prescription of EPA ethyl ester. Omega-3 fatty acids may reduce the risk of major cardiovascular events in patients with cardiovascular disease or risk factors, and reduce the risk of myocardial infarction in patients without cardiovascular disease; however, they may increase the risk of stroke in patients with myocardial infarction. In addition, prescription omega-3 acid ethyl ester has a good safety profile, and prescription EPA ethyl ester has a high risk of bleeding. Conclusion Moderate evidence showed that the use of omega-3 fatty acids may reduce the risk of major cardiovascular events, myocardial infarction, and cardiovascular death. Compared to other types of omega-3 fatty acids supplements, we support the use of prescription EPA ethyl ester formulations for the prevention of cardiovascular disease, but the potential risk of atrial fibrillation and bleeding cannot be ignored. It is important to note that omega-3 fatty acids should be applied with caution in patients with previous myocardial infarction, which may increase the risk of stroke. Finally, omega-3 fatty acids are relatively safe and in general do not increase gastrointestinal problems, bleeding-related disorders, or cancer, but attention needs to be paid to the risk of bleeding with prescription EPA ethyl ester formulations.

Background: The migration, proliferation and apoptosis of vascular smooth muscle cells (VSMCs) are critical for plaque stability. WNT-inducible signalling pathway protein-1 (WISP1), a member of the CCN family of extracellular matrix proteins, can expedite the migration and proliferation of VSMCs. However, its underlying mechanism and relationship with atherosclerosis remain elusive. The relationship between WISP1 and apoptosis of VSMCs has not been determined previously. Method: In the study, we aimed to investigate the relationship between WISP1 and plaque stability and its related mechanism. ApoE−/− mice were divided following groups: the null lentivirus (NC), lentivirus WISP1 (IvWISP1) and WISP1-shRNA (shWISP1) groups. Immunofluorescence, Oil Red O and Masson's staining of the carotid arteries were performed. Transwell wound healing assay, CCK8 assay, and TdT-mediated dUTP nick-end labeling (TUNEL) staining were performed using VSMCs. The levels of WISP1, P38, C-Jun N-terminal kinase, extracellular signal-regulated kinase (ERK), mitogen-activated extracellular signal-regulated kinase (MEK), focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI3K), Akt (also known as PKB, protein kinase B), mammalian target of rapamycin (mTOR), cleaved caspase3, Bcl2 and Bax were detected by western blotting. Results: The relative area of lipids and monocytes/macrophages in the shWISP1 group increased compared with that of the NC group. However, the relative area of smooth muscle cell and collagen in the IvWISP1 group increased compared with that in the NC group. Therefore, WISP1 could stabilize atherosclerotic plaques. Besides, WISP1 accelerate the migration and proliferation of VSMCs via integrin α5β1 and FAK/MEK/ERK signalling pathways. In addition, WISP1 can inhibit the apoptosis of VSMCs via the PI3K/Akt/mTOR pathway. Conclusion: WISP1 not only inhibits the apoptosis of VSMCs via the PI3K/Akt/mTOR pathway but also enhances the migration and proliferation of VSMCs via the integrin α5β1 and FAK/MEK/ERK pathways. Therefore, WISP1 could enhance the stability of atherosclerotic plaques.

In the past decade, direct oral anticoagulants (DOACs) have proven to be the best option for patients with nonvalvular atrial fibrillation. Nevertheless, evidence for the use of DOACs for anticoagulation in valvular atrial fibrillation, particularly after aortic valve replacement, remains inadequate. Thus, we conducted a meta‐analysis to compare the efficacy and safety of vitamin K antagonists (VKAs) and DOACs in patients with atrial fibrillation after transcatheter aortic valve replacement (TAVR). We conducted a comprehensive search of online databases, and 11 studies were included in the final analysis. The primary endpoint was all‐cause mortality. Secondary endpoints included stroke and cardiovascular death. The safe endpoint is major and/or life‐threatening bleeding. Subgroup analysis was conducted according to the different follow‐up time of each study. Random‐effects models were used for all outcomes. Statistical heterogeneity was assessed using χ2 tests and quantified using I2 statistics. Patients in the DOACs group had a significantly lower risk of all‐cause mortality compared with patients in the VKAs group (relative risk [RR]: 1.20, 95% confidence interval [CI]: 1.01–1.43, p = .04). This benefit may be greater with longer follow‐up. In a subgroup analysis based on the length of follow‐up, a significantly lower risk of all‐cause mortality was found in the DOACs group in the subgroup with a follow‐up time of >12 months (RR: 1.50, 95% CI: 1.07–2.09, p = .001). There were no significant differences between the two groups in cardiovascular death, stroke, and major and/or life‐threatening bleeding. For patients with atrial fibrillation after TAVR, the use of DOACs may be superior to VKAs, and the benefit may be greater with longer follow‐up. The anticoagulant strategy for atrial fibrillation after TAVR is a valuable direction for future research.

Diabetic cardiomyopathy (DCM) is one of the leading causes of heart failure in patients with diabetes mellitus, with limited effective treatments. The cardioprotective effects of sodium-glucose cotransporter 2(SGLT2) inhibitors have been supported by amounts of clinical trials, which largely fills the gap. However, the underlying mechanism still needs to be further explored, especially in terms of its protection against cardiac fibrosis, a crucial pathophysiological process during the development of DCM. Besides, endothelial-to-mesenchymal transition (EndMT) has been reported to play a pivotal role in fibroblast multiplication and cardiac fibrosis. This study aimed to evaluate the effect of SGLT2 inhibitor dapagliflozin (DAPA) on DCM especially for cardiac fibrosis and explore the underlying mechanism. In vivo, the model of type 2 diabetic rats was built with high-fat feeding and streptozotocin injection. Untreated diabetic rats showed cardiac dysfunction, increased myocardial fibrosis and EndMT, which was attenuated after treatment with DAPA and metformin. In vitro, HUVECs and primary cardiac fibroblasts were treated with DAPA and exposed to high glucose (HG). HG-induced EndMT in HUVECs and collagen secretion of fibroblasts were markedly inhibited by DAPA. Up-regulation of TGF-β/Smad signalling and activity inhibition of AMPKα were also reversed by DAPA treatment. Then, AMPKα siRNA and compound C abrogated the anti-EndMT effects of DAPA in HUVECs. From above all, our study implied that DAPA can protect against DCM and myocardial fibrosis through suppressing fibroblast activation and EndMT via AMPKα-mediated inhibition of TGF-β/Smad signalling.

OBJECTIVE:The purpose of the present study was to evaluate the role of co-treatment of rosuvastatin (RSV) and dapagliflozin (DGZ) preconditioning in myocardium ischemia/reperfusion (I/R) injury and to further investigate the underlying mechanism. METHODS:Sprague-Dawley (SD) rats (n = 25) were divided into five groups randomly: (1) Sham, (2) I/R, (3) I/R + RSV (10 mg/kg), (4) IR + DGZ (1 mg/kg), and (5) I/R + RSV (10 mg/kg) + DGZ (1 mg/kg). The I/R model was induced with 30 min of left anterior descending occlusion followed by 120 min of reperfusion. RESULTS:In vivo pretreatment with RSV and DGZ, respectively, showed a significant reduction of infarction size, a significant increase in the levels of left ventricular systolic pressure, and maximal rate increase in left ventricular pressure (+dp/dt max), decrease in the levels of left ventricular end-diastolic pressure (LVEDP), maximal rate of decrease of left ventricular pressure (-dp/dt max) and activity of cardiac enzymes of creatine kinase (CK), creatine kinase MB isoenzymes (CK-MB), and hyper-tensive cardiac troponin I compared with the I/R group. H9C2 cells were exposed to hypoxia/reoxygenation to simulate an I/R model. In vitro administration of 25 µM RSV and 50 µM DGZ significantly enhanced cell viability, upregulated the expression levels of p-PI3K, p-Akt, p-mTOR, and Bcl-2, whereas it downregulated cleaved-caspase3, Bax. TUNEL assay indicated that pretreatment with RSV and DGZ decreased the apoptosis of H9C2 cells. CONCLUSION:The combination of RSV and DGZ significantly enhances the cardioprotective effects compared with RSV or DGZ alone. RSV and DGZ have the potential cardioprotective effects against I/R injury by activating the PI3K/AKt/mTOR signaling pathway.

Lipid deposition in macrophages plays an important role in atherosclerosis. The WNT1‐inducible signalling pathway protein 1(WISP1) can promote proliferation and migration of smooth muscle cells. Its expression is up‐regulated in obesity, which is associated with atherosclerosis, but the effect of WISP1 on atherosclerosis remains unclear. Thus, the objective of our study was to elucidate the role of WISP and its mechanism of action in atherosclerosis via in vivo and in vitro experiments. In our experiment, ApoE‐/‐ mice were divided into 5 groups: control, high‐fat diet (HFD), null lentivirus (HFD + NC), lentivirus WISP1 (HFD + IvWISP1) and WISP1‐shRNA (HFD + shWISP1). Oil Red O staining, immunofluorescence and immunohistochemistry of the aortic sinuses were conducted. Macrophages (RAW264.7 cell lines and peritoneal macrophages) were stimulated with 50 μg/mL oxidized low‐density lipoprotein (ox‐LDL); then, the reactive oxygen species (ROS) level was measured. Oil Red O staining and Dil‐ox‐LDL (ox‐LDL with Dil dye) uptake measurements were used to test lipid deposition of peritoneal macrophages. WISP1, CD36, SR‐A and PPARγ expression levels were measured via Western blotting and ELISA. The results showed that HFD mice had increased WISP1, CD36 and SR‐A levels. The plaque lesion area increased when WISP1 was down‐regulated, and lipid uptake and foam cell formation were inhibited when WISP1 was up‐regulated. Treatment of RAW264.7 cell lines with ox‐LDL increased WISP1 expression via activation of the Wnt5a/β‐catenin pathway, whereas ROS inhibition reduced WISP1 expression. Moreover, WISP1 down‐regulated CD36 and SR‐A expression, and Oil Red O staining and Dil‐ox‐LDL uptake measurement showed that WISP1 down‐regulated lipid deposition in macrophages. These results clearly demonstrate that WISP1 is activated by ox‐LDL at high ROS levels and can alleviate lipid deposition in atherosclerosis through the PPARγ/CD36 pathway.

Background Pleckstrin homology (PH) domain leucine-rich repeat protein phosphatase 1 (PHLPP1) is a kind of serine/threonine phosphatase, whose dysregulation is accompanied with numerous human diseases. However, its role in diabetic cardiomyopathy remains unclear. We explored the underlying function and mechanism of PHLPP1 in diabetic cardiomyopathy (DCM). Method In vivo, Type 1 diabetic rats were induced by intraperitoneal injection of 60 mg/kg streptozotocin (STZ). Lentivirus-mediated short hairpin RNA (shRNA) was used to knock down the expression of PHLPP1. In vitro, primary neonatal rat cardiomyocytes and H9C2 cells were incubated in 5.5 mmol/L glucose (normal glucose, NG) or 33.3 mmol/L glucose (high glucose, HG). PHLPP1 expression was inhibited by PHLPP1-siRNA to probe into the function of PHLPP1 in high glucose-induced apoptosis in H9c2 cells. Results Diabetic rats showed up-regulated PHLPP1 expression, left ventricular dysfunction, increased myocardial apoptosis and fibrosis. PHLPP1 inhibition alleviated cardiac dysfunction. Additionally, PHLPP1 inhibition significantly reduced HG-induced apoptosis and restored PI3K/AKT/mTOR pathway activity in H9c2 cells. Furthermore, pretreatment with LY294002, an inhibitor of PI3K/Akt/mTOR pathway, abolished the anti-apoptotic effect of PHLPP1 inhibition. Conclusion Our study indicated that PHLPP1 inhibition alleviated cardiac dysfunction via activating the PI3K/Akt/mTOR signalling pathway in DCM. Therefore, PHLPP1 may be a novel therapeutic target for human DCM.

Nicorandil exerts myocardial protection through its antihypoxia and antioxidant effects. Here, we investigated whether it plays an anti‐apoptotic role in diabetic cardiomyopathy. Sprague‐Dawley rats were fed with high‐fat diet; then single intraperitoneal injection of streptozotocin was performed. Rats with fasting blood glucose (FBG) higher than 11.1 mmol/L were selected as models. Eight weeks after the models were built, rats were treated with nicorandil (7.5 mg/kg day and 15 mg/kg day respectively) for 4 weeks. H9c2 cardiomyocytes were treated with nicorandil and then stimulated with high glucose (33.3 mmol/L). TUNEL assay and level of bcl‐2, bax and caspase‐3 were measured. 5‐HD was used to inhibit nicorandil. Also, PI3K inhibitor (Miltefosine) and mTOR inhibitor (rapamycin) were used to inhibit PI3K/Akt pathway. The results revealed that nicorandil (both 7.5 mg/kg day and 15mg/kg day) treatment can increase the level of NO in the serum and eNOS in the heart of diabetic rats compared with the untreated diabetic group. Nicorandil can also improve relieve cardiac dysfunction and reduce the level of apoptosis. In vitro experiments, nicorandil (100 µmol) can attenuate the level of apoptosis stimulated by high glucose significantly in H9C2 cardiomyocyte compared with the untreated group. The effect of nicorandil on apoptosis was blocked by 5‐HD, and it was accompanied with inhibition of the phosphorylation of PI3K, Akt, eNOS, and mTOR. After inhibition of PI3K/Akt pathway, the protective effect of nicorandil is restrained. These results verified that as a NO donor, nicorandil can also inhibit apoptosis in diabetic cardiomyopathy which is mediated by PI3K/Akt pathway.

AIMS:Absent in melanoma 2 (AIM2) is a cytosolic DNA sensor which plays an important role in inflammasome formation and is involved in various cellular functions including pyroptosis, fibrosis, and tissue injury. Our study aimed to investigate whether AIM2 plays a role in diabetic cardiomyopathy (DCM) and to explore its potential molecular mechanism. MAIN METHODS:Sprague-Dawley rats were randomly divided into 4 groups: Control, Diabetes Mellitus (DM), DM + shAIM2, and DM + shNC. The cardiac function of rats was measured. Hematoxylin and eosin staining, Masson's staining, sinus red staining, and immunohistochemistry were performed. H9c2 cardiomyocytes were cultured in DMEM and stimulated with high-glucose treatment (25 mmol/l). The level of reactive oxygen species (ROS) was measured. AIM2-siRNA were used to inhibit the expression of AIM2. TUNEL assay and EthD-III staining were used to measure cell death. The expression levels of AIM2, ASC, caspase-1, IL-1β, and GSDMD-N were measured by western blotting. KEY FINDINGS:In the streptozotocin-induced diabetic rat model, AIM2 expression was significantly increased in heart tissue compared with the control. Also, diabetic rats exhibited severe left ventricular dysfunction including metabolic disorder, cardiac fibrosis, and cardiomyocyte death. Gene silencing of AIM2 alleviated cardiac dysfunction which resulted from metabolic disorder and ventricular remodelling. In vitro, treatment of H9C2 cardiomyoblasts with HG significantly increased AIM2, while ROS inhibition reduced the level of AIM2. AIM2-siRNA alleviated GSDMD-N-related pyroptosis in H9c2 cardiomyoblasts. SIGNIFICANCE:Our results indicate that AIM2 plays an important role in cell death and fibrosis in HG-induced, ROS-mediated diabetic cardiomyopathy via the GSDMD pathway.

Endometrial cancer is the most common cancer of the female reproductive system in the developed countries. Metformin is a widely used medication that has been prescribed to treat type 2 diabetes. In recent years, metformin has been found to improve the survival prognosis of cancer patients clinically. We aimed to investigate inhibition of metformin on the proliferation of endometrial carcinoma. Metformin was used to treat endometrial cancer cell lines Ishikawa and RL95-2. The expression of programmed death-ligand 1 (PD-L1) in the treated cells was assessed by western blot. The tumor cell proliferation was evaluated by colony formation assay. The binding between PD-L1 and AMP-activated protein kinase (AMPK) was identified by co-immunoprecipitation. Ishikawa and RL95-2 cells were co-cultured with activated T cells to detect the survival of Ishikawa and RL95-2 cells in the presence or absence of metformin. Our results showed that metformin treatment on endometrial cancer cells Ishikawa and RL95-2 decreased the expression level of PD-L1 protein. Metformin treatment significantly activated T cells against Ishikawa and RL95-2 cells. We demonstrated that the inhibition of PD-L1 by metformin is dependent on the AMPK signaling protein, and that metformin promotes direct binding of the AMPK protein to the PD-L1 protein. We confirmed that metformin, a conventional medication used in diabetes therapy, holds anti-tumor activity in endometrial cancer. The suppression of metformin is relevant to the inhibition of PD-L1 expression and the activation of AMPK signaling protein, providing a novel mechanism in the anti-tumor property of metformin.

OBJECTIVESTo analyse the effect of dydrogesterone use during pregnancy on uterine fibroids, pregnancy complications, and pregnancy outcome.MATERIAL AND METHODSIn all, 372 pregnant women with uterine fibroids who were treated at the Affiliated Provincial Hospital of Shandong University were included in this study. Thirty-three of these women received dydrogesterone and constituted the treatment group, and the 27 women who were found to have uterine fibroids during the first trimester but did not receive intervention to prevent miscarriage composed the control group. The changes in uterine fibroids before and after pregnancy and the pregnancy complications were recorded; immunohistochemistry was used to detect the expression of progesterone receptor (PR) and proliferation- and apoptosis-related proteins in the uterine fibroid tissue.RESULTSNo significant difference was observed in the change in uterine fibroid volume during pregnancy between the treatment group and the control group (p > 0.05). The percentage of uterine fibroids with red degeneration was lower in the treatment group than in the control group, but the difference was not statistically significant. No significant difference was observed in newborn weight, height, Apgar score, threatened miscarriage, or premature birth, among other characteristics, between the two groups (p > 0.05). Immunohistochemistry showed no significant difference in the expression of PR, cyclinD1, insulin-like growth factor (IGF1), or B-cell lymphoma 2 (Bcl2) between the two groups.CONCLUSIONSThe use of dydrogesterone during pregnancy has no significant effect on uterine fibroids, pregnancy progression, or pregnancy outcomes in pregnant patients with uterine fibroids.

Adenomyosis is a finding that is associated with dysmenorrhea and heavy menstrual bleeding, associated with PI3K/AKT signaling overactivity. To investigate the effect of metformin on the growth of eutopic endometrial stromal cells (ESCs) from patients with adenomyosis and to explore the involvement of AMP-activated protein kinase (AMPK) and PI3K/AKT pathways. Primary cultures of human ESCs were derived from normal endometrium (normal endometrial stromal cells (N-ESCs)) and adenomyotic eutopic endometrium (adenomyotic endometrial stroma cells (A-ESCs)). Expression of AMPK was determined using immunocytochemistry and western blot analysis. 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assays were used to determine the effects of metformin and compound C on ESCs and also to detect growth and proliferation of ESCs. AMPK and PI3K/AKT signaling was determined by western blotting. A-ECSs exhibited greater AMPK expression than N-ESCs. Metformin inhibited proliferation of ESCs in a concentration-dependent manner. The IC50 was 2.45 mmol/l for A-ESCs and 7.87 mmol/l for N-ESCs. Metformin increased AMPK activation levels (p-AMPK/AMPK) by 2.0±0.3-fold in A-ESCs, 2.3-fold in A-ESCs from the secretory phase, and 1.6-fold in the proliferation phase. The average reduction ratio of 17β-estradiol on A-ESCs was 2.1±0.8-fold in proliferative phase and 2.5±0.5-fold in secretory phase relative to the equivalent groups not treated with 17β-estradiol. The inhibitory effects of metformin on AKT activation (p-AKT/AKT) were more pronounced in A-ESCs from the secretory phase (3.2-fold inhibition vs control) than in those from the proliferation phase (2.3-fold inhibition vs control). Compound C, a selective AMPK inhibitor, abolished the effects of metformin on cell growth and PI3K/AKT signaling. Metformin inhibits cell growth via AMPK activation and subsequent inhibition of PI3K/AKT signaling in A-ESCs, particularly during the secretory phase, suggesting a greater effect of metformin on A-ESCs from secretory phase.