Forkhead Box Protein O1 Research Articles

Circ_BLNK is a Unique Molecular Marker in Non-small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) patients are characterized by distant metastasis and poor prognosis. Growing evidence has implied that circular RNAs (circRNAs) are involved in multiple tumor progression, including NSCLC. The objective of the present study was to functionally dissect the role and mechanism of circ_BLNK in NSCLC development and progression. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression of circ_BLNK, miR-942-5p, and forkhead box protein O1 (FOXO1) in NSCLC tissues and cells. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, 5-ethynyl-2'-deoxyuridine (EdU) assay and colony formation assay detected cell proliferation; the protein expression levels were tested by western blot assay; cell apoptosis was measured by flow cytometry, and transwell assay detected cell migration and invasion. The molecular targeting relationship was determined by dual-luciferase reporter assay. The effect of circ_BLNK overexpression on tumor growth was detected by in vivo experiments and immunohistochemistry. Circ_BLNK was dramatically decreased in NSCLC, and overexpression of circ_BLNK inhibited proliferation, migration, and invasion of NSCLC cells and promoted cell apoptosis. Circ_BLNK level was negatively correlated with miR-942-5p expression and positively correlated with FOXO1 expression. Moreover, circ_BLNK acted as a sponge for miR-942-5p, which targeted FOXO1. Rescue assays presented that miR-942-5p reversed the anticancer action of circ_BLNK in NSCLC. Besides that, miR-942-5p inhibition suppressed the oncogenic behaviors, which were attenuated by FOXO1 knockdown. Animal experiments exhibited that circ_BLNK upregulation repressed tumor growth in vivo. Our study demonstrated a novel regulatory mechanism that circ_BLNK/miR-942-5p/FOXO1 axis adjusted non-small cell lung cancer development.

FoxO1 promotes ovarian cancer by increasing transcription and METTL14-mediated m6A modification of SMC4.

The transcription factor forkhead box protein O1 (FoxO1) is closely related to the occurrence and development of ovarian cancer (OC), however its role and molecular mechanisms remain unclear. Herein, we found that FoxO1 was highly expressed in clinical samples of OC patients and was significantly correlated with poor prognosis. FoxO1 knockdown inhibited the proliferation of OC cells invitro and invivo. ChIP-seq combined with GEPIA2 and Kaplan-Meier database analysis showed that structural maintenance of chromosome 4 (SMC4) is a downstream target of FoxO1, and FoxO1 promotes SMC4 transcription by binding to its -1400/-1390 bp promoter. The high expression of SMC4 significantly blocked the tumor inhibition effect of FoxO1 knockdown. Furtherly, FoxO1 increased SMC4 mRNA abundance by transcriptionally activating methyltransferase-like 14 (METTL14) and increasing SMC4 m6A methylation on its coding sequence region. The Cancer Genome Atlas dataset analysis confirmed a significant positive correlation between FoxO1, SMC4, and METTL14 expression in OC. In summary, this study revealed the molecular mechanisms of FoxO1 regulating SMC4 and established a clinical link between the expression of FoxO1/METTL14/SMC4 in the occurrence of OC, thus providing a potential diagnostic target and therapeutic strategy.

FOXO3a Induces Myocardial Fibrosis by Upregulating Mitophagy.

Atrial fibrillation is one of the most common cardiac arrhythmias. Myocardial fibrosis is closely associated with atrial remodeling, which leads to heightened risk of atrial fibrillation. This study aimed to explore whether forkhead box protein O3 (FOXO3a) impacts myocardial fibrosis incidence by regulating mitophagy. Cell viability was assessed by cell counting kit-8 (CCK-8) assays. The expression of vimentin and cytochrome C was detected by immunofluorescence assays. Quantitative real-time polymerase chain reaction (PCR) was used to analyze the relative mRNA level of FOXO3a. Expression of FOXO3a, phosphorylated FOXO3a, Collagen I, Collagen III, alpha-smooth muscle actin (α-SMA), matrix metalloprotease 9 (MMP9), light chain 3 (LC3), phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1), Parkin, and sequestosome-1 (p62) proteins were determined by western blotting. 5-ethynyl-2'-deoxyuridine (EDU) incorporation was employed to measure cell proliferation. Changes in mitochondrial membrane potential were determined by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide (JC-1) staining. A wound healing assay was used to examine cell migration, and the levels of reactive oxygen species were determined by flow cytometry. The expression of FOXO3a was upregulated in cardiac fibroblasts treated with angiotensin II (AngII), while the expression of phosphorylated FOXO3a was downregulated under these conditions. FOXO3a knockdown significantly inhibited the proliferation, migration and collagen secretion of cardiac fibroblasts treated with AngII. The ratio of LC3 II/I as well as expression of PINK1 and Parkin was increased, and the expression of p62 was decreased, in cardiac fibroblasts treated with AngII. Moreover, these effects were limited by FOXO3a knockdown. Finally, the mitophagy inducer everolimus (RAD001) attenuated the suppressive effect of FOXO3a knockdown on cardiac fibroblast activation. FOXO3a promotes the progress of myocardial fibrosis by triggering mitophagy in cardiac fibroblasts.

The Roles and Regulatory Mechanisms of Tight Junction Protein Cingulin and Transcription Factor Forkhead Box Protein O1 in Human Lung Adenocarcinoma A549 Cells and Normal Lung Epithelial Cells.

Tight junction (TJ) protein cingulin (CGN) and transcription factor forkhead box protein O1 (FOXO1) contribute to the development of various cancers. Histone deacetylase (HDAC) inhibitors have a potential therapeutic role for some cancers. HDAC inhibitors affect the expression of both CGN and FOXO1. However, the roles and regulatory mechanisms of CGN and FOXO1 are unknown in non-small cell lung cancer (NSCLC) and normal human lung epithelial (HLE) cells. In the present study, to investigate the effects of CGN and FOXO1 on the malignancy of NSCLC, we used A549 cells as human lung adenocarcinoma and primary human lung epithelial (HLE) cells as normal lung tissues and performed the knockdown of CGN and FOXO1 by siRNAs. Furthermore, to investigate the detailed mechanisms in the antitumor effects of HDAC inhibitors for NSCLC via CGN and FOXO1, A549 cells and HLE cells were treated with the HDAC inhibitors trichostatin A (TSA) and Quisinostat (JNJ-2648158). In A549 cells, the knockdown of CGN increased bicellular TJ protein claudin-2 (CLDN-2) via mitogen-activated protein kinase/adenosine monophosphate-activated protein kinase (MAPK/AMPK) pathways and induced cell migration, while the knockdown of FOXO1 increased claudin-4 (CLDN-4), decreased CGN, and induced cell proliferation. The knockdown of CGN and FOXO1 induced cell metabolism in A549 cells. TSA and Quisinostat increased CGN and tricellular TJ protein angulin-1/lipolysis-stimulated lipoprotein receptor (LSR) in A549. In normal HLE cells, the knockdown of CGN and FOXO1 increased CLDN-4, while HDAC inhibitors increased CGN and CLDN-4. In conclusion, the knockdown of CGN via FOXO1 contributes to the malignancy of NSCLC. Both HDAC inhibitors, TSA and Quisinostat, may have potential for use in therapy for lung adenocarcinoma via changes in the expression of CGN and FOXO1.

Remedial effects of tilapia skin peptides against dexamethasone-induced muscle atrophy in mice by modulation of AKT/FOXO3a and Sirt1/PGC-1α signaling pathways

Tilapia skin peptides (TSP) possess a range of physiological activities. This study aimed to explore the effects of TSP on Dexamethasone (DEX)-induced muscle atrophy. In vitro, C2C12 myotube myotube diameter and expression levels of the muscle-specific E3 ubiquitin ligases F-box only protein 32 (Atrogin-1) and muscle ring finger protein 1 (MuRF1) challenged with DEX were reversed by TSP. In vivo, DEX was injected subcutaneously to build muscle atrophy model mice. TSP enhanced grip strength, running distance, body lean muscle content, cross-sectional area of the gastrocnemius and tibialis anterior muscles of DEX-induced mice. Moreover, TSP inhibited the expression levels of Atrogin-1 and MuRF1. Mechanically, TSP improved DEX-induced muscle atrophy by regulating the Protein Kinase B α (AKT)/Forkhead box O3 protein (FOXO3a), Sirtuin 1 (Sirt1)/peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) signaling pathway, and downstream factors such as nuclear respiratory factor (NRF)1/2 and mitochondrial transcription factor A (TFAM).

Insulin-like growth factor-1 reduces cardiac autosis through decreasing AMPK/FOXO1 signaling and Na+/K+-ATPase-Beclin-1 interaction.

Insulin-like growth factor-1 (IGF-1) promotes survival and inhibits cardiac autophagy disruption. Male Wistar rats were treated with IGF-1 (50 µg/kg), and 24 h after injection hearts were excised. The level of interaction between Beclin-1 and the α1 subunit of sodium/potassium-adenosine triphosphates (Na+/K+-ATPase), and phosphorylated forms of IGF-1 receptor/insulin receptor (IGF-1R/IR), forkhead box protein O1 (FOXO1) and AMP-activated protein kinase (AMPK) were measured. The results indicate that IGF-1 decreased Beclin-1's association with Na+/K+-ATPase (p < 0.05), increased IGF-1R/IR and FOXO1 phosphorylation (p < 0.05), and decreased AMPK phosphorylation (p < 0.01) in rats' hearts. The new IGF-1 therapy may control autosis and minimize cardiomyocyte mortality.

O-GlcNAcylation mediates H2O2-induced apoptosis through regulation of STAT3 and FOXO1.

The O-linked-β-N-acetylglucosamine (O-GlcNAc) glycosylation (O-GlcNAcylation) is a critical post-translational modification that couples the external stimuli to intracellular signal transduction networks. However, the critical protein targets of O-GlcNAcylation in oxidative stress-induced apoptosis remain to be elucidated. Here, we show that treatment with H2O2 inhibited O-GlcNAcylation, impaired cell viability, increased the cleaved caspase 3 and accelerated apoptosis of neuroblastoma N2a cells. The O-GlcNAc transferase (OGT) inhibitor OSMI-1 or the O-GlcNAcase (OGA) inhibitor Thiamet-G enhanced or inhibited H2O2-induced apoptosis, respectively. The total and phosphorylated protein levels, as well as the promoter activities of signal transducer and activator of transcription factor3 (STAT3) and Forkhead box protein O 1 (FOXO1) were suppressed by OSMI-1. In contrast, overexpressing OGT or treating with Thiamet-G increased the total protein levels of STAT3 and FOXO1. Overexpression of STAT3 or FOXO1 abolished OSMI-1-induced apoptosis. Whereas the anti-apoptotic effect of OGT and Thiamet-G in H2O2-treated cells was abolished by either downregulating the expression or activity of endogenous STAT3 or FOXO1. These results suggest that STAT3 or FOXO1 are the potential targets of O-GlcNAcylation involved in the H2O2-induced apoptosis of N2a cells.

Metadichol® induced expression of Sirtuin family 1-7 in somatic and cancer cells

The Sirtuins 1-7 family and Klotho (KL), Forkhead box protein O1 (FOXO1), telomerase reverse transcriptase (TERT), tumor suppressor p53 (TP53) and growth differentiation factor 11 (GDF11) regulate aging, metabolism, and DNA repair and are involved in age-related diseases such as cancer, cardiovascular disease, and diabetes. Seven sirtuin genes in humans encode seven sirtuin enzymes (SIRT1–7), each of which has unique functions and subcellular locations. Sirtuins are nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylases that play a significant role in physiological processes such as energy metabolism, stress responses, DNA repair, and gene expression and are potential targets for age-related diseases such as type 2 diabetes, inflammatory diseases, and neurodegenerative disorders. They also play a role in cancer by regulating critical cellular processes such as DNA repair and energy metabolism. Other genes, such as Klotho (KL), Forkhead box protein O1 (FOXO1), telomerase reverse transcriptase (TERT), tumor suppressor p53 (TP53) and growth differentiation factor 11 (GDF11), also regulate aging, metabolism, and DNA repair and are involved in age-related diseases such as cancer, cardiovascular disease, and diabetes. In addition, these proteins are closely related to sirtuins. A single molecule that can activate these five genes and sirtuin genes is challenging because each isoform has a unique structure, substrate, and regulatory mechanism. Most known sirtuin activators are specific for Sirtuin 1, the most studied isoform of the sirtuin family. This study was initiated based on previous work in which we showed that metadichol can express all nuclear receptors if it is possible to express all seven sirtuin families 1-7 using metadichol as a small molecule inducer. Herein, we report that at concentrations ranging from 1 pg/mL to 100 ng/mL, Metadichol®, a nanoemulsion of long-chain alcohols, induced the expression of the human Sirtuin 1-7 gene in dermal fibroblasts and a variety of cancer cells in a concentration-dependent manner and that KL, GDF11, telomerase, Foxo1 and P53 could have significant beneficial effects on mitigating age-related diseases. The results were quantified by using qRT‒PCR, and proteins were characterized using western blot techniques. The experimental procedure used is unique in that it did not involve the use of viruses or other gene insertion technique.

Homeobox B2 promotes malignant behavior and contributes to the radioresistance of nasopharyngeal carcinoma by regulating forkhead box protein O1.

Background: Nasopharyngeal carcinoma (NPC) is an epithelial tumor of the head and neck with heterogeneous racial and geographical distributions. Homeobox B2 (HOXB2) is a tumor promoter in many cancers. However, the biological role of HOXB2 in NPC has not been elucidated. Methods: Bioinformatics analysis was performed to identify the differentially expressed genes (DEGs) between samples of patients with radiosensitive and radioresistant NPC. qRT-PCR, western blotting and immunohistochemistry were used to detect the expression levels of the corresponding mRNA and proteins. Cell viability was detected by CCK-8 assay and colony-forming capability was evaluated using colony formation assays. Further, migration and invasion abilities were examined using wound-healing and transwell chamber assays, respectively. Cellular apoptosis after irradiation was assessed using flow cytometry and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. Results: HOXB2 was identified as a potential regulator of radioresistance in NPC. Our in vitro results indicate that HOXB2 overexpression (HOXB2-OE) promoted malignant behaviors including invasion, migration, proliferation, and inhibited the irradiation-induced apoptosis of NPC cells. Consistent with these results, HOXB2 knockdown (HOXB2-sh) exhibited the opposite trends in these biological activities. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the DEGs were enriched in the FOXO signaling pathway. Mechanistically, western blotting showed that HOXB2-OE inhibited forkhead box protein O1 (FOXO1) expression in NPC cells. Thereafter, we transferred the FOXO1-OE plasmid to HOXB2-OE NPC cells and found that overexpression of FOXO1 reversed cell proliferation, migration, invasion, and radioresistance profiles promoted by HOXB2 overexpression. Conclusion: Our findings showed that HOXB2 acts as a tumor promoter in NPC, activating malignant behaviors and radioresistance of tumors via FOXO1 regulation. Moreover, the inactivation of HOXB2 or activation of FOXO1 are potential strategies to inhibit tumor progression and overcome radioresistance in NPC.

Zingerone attenuates sciatic nerve damage caused by sodium arsenite by inhibiting NF-κB, caspase-3, and ATF-6/CHOP pathways and activating the Akt2/FOXO1 pathway.

In the present study, the potential protective effects of zingerone (ZNG) against sciatic nerve damage caused by sodium arsenite (SA), a common environmental pollutant, were evaluated by various biochemical, molecular, and histological methods. In the study, SA and ZNG were given to 35 male Sprague Dawley rats for 14 days. At the end of the period, the sciatic nerve tissues were taken and the markers involved in oxidative stress, endoplasmic reticulum stress, inflammation, and apoptosis were analyzed. The data obtained showed that SA decreased glutathione (GSH) levels and increased malondialdehyde (MDA) levels in the sciatic nerve tissue. However, it was determined that these markers approached the control group levels due to the anti-oxidant properties of ZNG. While SA triggered endoplasmic reticulum stress and apoptosis pathways, ZNG suppressed them. Moreover, SA up-regulated inflammatory markers such as nuclear factor kappa-B (NF-κB), tumor necrosis factor-alpha (TNF-α), interleukin-1-beta (IL-1β), and neuronal nitric oxide synthases (nNOS) in the sciatic nerves and caused neuro-inflammation and inhibited cell survival by suppressing serine/threonine-protein kinase 2 (Akt2) and forkhead box protein O1 (FOXO1) genes. It has also been shown histopathologically that SA causes degeneration in the sciatic nerves. In contrast, ZNG suppressed neuro-inflammation, activated Akt2/FOXO1 signaling, and repaired histological irregularities. In general, SA caused oxidative stress, inflammation, ER stress, and apoptosis in the sciatic nerves of rats, causing damage to the tissues, however, ZNG suppressed these pathways and protected the sciatic nerves from the destructive effect of SA.

Blockade of angiotensin II modulates insulin-like growth factor 1-mediated skeletal muscle homeostasis in experimental steatohepatitis

Sarcopenia is associated with mortality in patients with nonalcoholic steatohepatitis (NASH). Angiotensin II receptor blocker (ARB) has been suggested to prevent sarcopenia, but reports on its effect on NASH-derived skeletal muscle atrophy in conjunction with insulin-like growth factor 1 (IGF-1)-mediated muscle homeostasis are few. Our aim was to examine the combined effect of the ARB losartan and IGF-1 replacement on skeletal muscle atrophy in a methionine–choline deficient (MCD) diet-fed murine steatohepatitis model. The MCD-fed mice developed steatohepatitis and skeletal muscle atrophy, as indicated by the reduction of psoas muscle mass and attenuation of forelimb and hindlimb grip strength. Significantly suppressed steatohepatitis and skeletal muscle atrophy was observed after single treatment with ARB or IGF-1, and these effects were augmented after combination treatment. Treatment with ARB and IGF-1 effectively inhibited ubiquitin proteasome-mediated protein degradation by reducing forkhead box protein O1 (FOXO1) and FOXO3a transcriptional activity in the skeletal muscle. Combined ARB and IGF-1 decreased the intramuscular expression of proinflammatory cytokines (i.e., TNFα, IL6, and IL1β) and increased the Trolox equivalent antioxidant capacity and antioxidant enzymes (CAT, GPX1, SOD2, and CYTB). This antioxidant effect was based on downregulation of NADPH oxidase (NOX) 2, normalization of mitochondrial biogenesis and dynamics. Moreover, ARB increased the hepatic and plasma IGF-1 levels and improved steatohepatitis, leading to enhanced skeletal muscle protein synthesis mediated by IGF-1/ AKT/ mechanistic target of rapamycin signaling. Collectively, combined ARB and IGF-1 replacement could be a promising new therapeutic target for NASH-derived skeletal muscle wasting.

Tumor-derived exosomal miR-1247-3p promotes angiogenesis in bladder cancer by targeting FOXO1

ABSTRACTTumor-derived exosomes are highly correlated with tumor progression and angiogenesis. This study was designed to probe the role of tumor-derived exosomal miR-1247-3p in mediating the angiogenesis in bladder cancer. Exosomes isolation from the culture medium of normal or bladder cancer cell lines was performed using a differential centrifugation method. miR-1247-3p expression in exosomes and cells was detected by quantitative real-time PCR (qRT-PCR). The effect of exosomes on the angiogenesis of human umbilical vein endothelial cells (HUVECs) was assessed using cell counting kit-8 (CCK-8), transwell and tube formation assays. The interaction between miR-1247-3p and forkhead box protein O1 (FOXO1) was studied using luciferase reporter and RNA pull down assays. Exosomes were successfully isolated from T24, UM-UC-3, and SV-HUC-1 cells, as confirmed by corresponding identifications. Functional experiments revealed that exosomes derived from T24 and UM-UC-3 cells significantly enhanced the abilities of proliferation, migration, angiogenesis, and vascular endothelial-derived growth factor (VEGF) secretion in HUVECs. miR-1247-3p was highly expressed in exosomes derived from T24 and UM-UC-3 cells, and exosomes derived from miR-1247-3p inhibitor-transfected cells reduced HUVEC viability, migration, tube formation, and VEGF level. FOXO1 was confirmed as a direct target of miR-1247-3p. Rescue assays suggested that the effect of miR-1247-3p inhibition on the viability, migration, and angiogenesis of HUVECs was partly abrogated by the knockdown of FOXO1. Our data suggest that miR-1247-3p is up-regulated in tumor-derived exosomes, thereby inhibiting FOXO1 expression and facilitating angiogenesis in bladder cancer.

Bioinformatics procedure for investigating senolytic (anti-aging) agents: A digital signal processing technique.

Cell growth involves cell division. This stops after reaching a certain limit. Some cells become inactive and unable to undergo apoptosis (programmed cell death). These cells accumulate at sites of tissue damage or disease, thus accelerating aging. They are called senescent cells. Therapeutic interventions that can either eliminate senescent cells (senolytics) or suppress their harmful effects (senomorphics) have been developed. Senescence (aging) is caused by the inter- and intramolecular interactions between the domains of forkhead (FHD) and transactivation (TAD), as well as C-terminal region 3 (CR3) and DNA binding (DBD). On the other hand, anti-senescent/senolytic (anti-aging) activities are achieved by disrupting these interactions with CR3- and forkhead box protein O4 (FOXO4)-based peptides, such as ES2 and DRI, respectively. In this study, we use a computerized procedure based on digital signal processing to systematically analyze the inter-molecular interactions between senolytics and their targets. Informational spectrum method (ISM) is engaged. We obtained the sequences of the peptides from the interacting proteins of CR3 and FOXO4 and evaluated their ability to disrupt the inter-molecular interactions between FOXO4 and DRI and CR3 and BDB, which are responsible for senescence (aging). Our results show that the peptides have different degrees of senolytic (anti-aging) activity, depending on their affinity for CR3 and BDB, or FOXO4 and DRI. We found that enhanced senescence 2 (ES2) has a higher affinity for CR3 and BDB than FOXO4 and DRI, and that the interaction between CR3 and BDB is crucial for aging. Therefore, ES2 and other CR3-based peptides are more potent senolytics than FOXO4-based peptides. Our findings are consistent with previous studies and reveal new insights into the mechanisms of senescence and senolytics. ES2 is considered the best senolytic candidate, as it is 3-7 times more effective than DRI. We verified that ES2 has a weaker interaction with FOXO4 than CR3. However, the performance of DRI has been noted to depend on its intramolecular interactions and stability. Hence, intramolecular analyses using the digital signal processing-based technique has become very vital and will follow. CR3-based peptides are promising candidates for senolytic therapy. Senolytics are linear chains of amino acids that can target and eliminate senescent cells, which are cells that have stopped dividing and contribute to aging and age-related diseases. By using this proposed, novel computerized technique that is based on digital signal processing, senolytics can be easily analyzed and optimized for their effectiveness and safety. This provides a more rational approach to enhancing our longevity and well-being by offering interventions that can delay or reverse aging and insights that can advance the field of gerontology. This procedure also will compliment other approaches such as molecular stimulation, etc.

Gandouling alleviates nerve injury through PI3K/Akt/FoxO1 and Sirt1/FoxO1 signaling pathway to inhibit autophagy in the rats model of Wilson's disease.

Previous studies have shown that Gandouling (GDL) may alleviate the nerve damage caused by Wilson's disease (WD) by inhibiting the autophagy of nerve cell mitochondria. However, its mechanisms are still unclear. Revealing the therapeutic mechanism of GDL is beneficial for its clinical application and provides theoretical support for the development of new formulations for treating WD. This time we found that the oxidative stress level in the body of the copper-overloaded WD rates increased, neurons in the hippocampus were damaged, and autophagy occurred. GDL reversed these situations and significantly improved the learning, memory, and spatial cognitive abilities of the high-copper-loaded WD rates. After GDL intervention, the expression of phosphatidylinositol-3 kinase (PI3K), phosphorylated serine-threonine protein kinase (AKT), and phosphorylated forkhead box protein O1 (FoxO1) significantly increased, whereas FoxO1 in the nucleus decreased and phosphorylated FoxO1 in the cytoplasm also significantly raised. In addition, the expression of Sirt1 significantly declined, and Ac-FoxO1 in the nucleus also significantly increased. These data indicated that GDL may promote the phosphorylation of FoxO1 and promote its nucleation by activating the PI3K/AKT/FoxO1 signaling pathway and inhibit Ac-FoxO1 hydrolysis in the nucleus through the Sirt1/FoxO1 signaling pathway to suppress the transcriptional activity of FoxO1. Furthermore, it inhibited the expression of autophagy genes Atg12 and Gabarapl1. In summary, our work provides new insights into the potential mechanisms of GDL repairing WD neuronal damage through autophagy pathways.

Elucidation of the anti-β-cell dedifferentiation mechanism of a modified Da Chaihu Decoction by an integrative approach of network pharmacology and experimental verification

Ethnopharmacological relevanceModified Da Chaihu decoction (MDCH) is a traditional Chinese herbal prescription that has been used in the clinic to treat type 2 diabetes (T2D). Previous studies have confirmed that MDCH improves glycemic and lipid metabolism, enhances pancreatic function, and alleviates insulin resistance in patients with T2D and diabetic rats. Evidence has demonstrated that MDCH protects pancreatic β cells via regulating the gene expression of sirtuin 1 (SIRT1) and forkhead box protein O1 (FOXO1). However, the detailed mechanism remains unclear. Aim of the studyDedifferentiation of pancreatic β cells mediated by FOXO1 has been recognized as the main pathogenesis of T2D. This study aims to investigate the therapeutic effects of MDCH on T2D in vitro and in vivo to elucidate the potential molecular mechanisms. Materials and methodsTo predict the key targets of MDCH in treating T2D, network pharmacology methods were used. A T2D model was induced in diet-induced obese (DIO) C57BL/6 mice with a single intraperitoneal injection of streptozotocin. Glucose metabolism indicators (oral glucose tolerance test, insulin tolerance test), lipid metabolism indicators (total cholesterol, triglyceride, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol), inflammatory factors (C-reactive protein, interleukin 6, tumor necrosis factor alpha), oxidative stress indicators (total antioxidant capacity, superoxide dismutase, malondialdehyde), and hematoxylin and eosin staining were analyzed to evaluate the therapeutic effect of MDCH on T2D. Immunofluorescence staining and quantification of FOXO1, pancreatic and duodenal homeobox 1 (PDX1), NK6 homeobox 1 (NKX6.1), octamer-binding protein 4 (OCT4), neurogenin 3 (Ngn3), insulin, and SIRT1, and Western blot analysis of insulin, SIRT1, and FOXO1 were performed to investigate the mechanism by which MDCH inhibited pancreatic β-cell dedifferentiation. ResultsThe chemical ingredients identified in MDCH were predicted to be important for signaling pathways related to lipid metabolism and insulin resistance, including lipids in atherosclerosis, the advanced glycation end product receptor of the advanced glycation end product signaling pathway, and the FOXO signaling pathway. Experimental studies showed that MDCH improved glucose and lipid metabolism in T2D mice, alleviated inflammation and oxidative stress damage, and reduced pancreatic pathological damage. Furthermore, MDCH upregulated the expression levels of SIRT1, FOXO1, PDX1, and NKX6.1, while downregulating the expression levels of OCT4 and Ngn3, which indicated that MDCH inhibited pancreatic dedifferentiation of β cells. ConclusionsMDCH has therapeutic effects on T2D, through regulating the SIRT1/FOXO1 signaling pathway to inhibit pancreatic β-cell dedifferentiation, which has not been reported previously.

Klotho inhibits IGF1R/PI3K/AKT signalling pathway and protects the heart from oxidative stress during ischemia/reperfusion injury

Ischemia/reperfusion injury (IRI) of the heart involves the activation of oxidative and proapoptotic pathways. Simultaneously Klotho protein presents anti-aging, antiapoptotic and antioxidative properties. Therefore, this study aimed to evaluate the effect of Klotho protein on oxidative stress in hearts subjected to IRI. Isolated rat hearts perfused with the Langendorff method were subjected to ischemia, followed by reperfusion, in the presence or absence of recombinant rat Klotho protein. The factors involved in the activation of insulin-like growth factor receptor (IGF1R)/phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) signalling pathway were evaluated. IRI caused activation of the IGF1R (p = 0.0122)/PI3K (p = 0.0022) signalling, as compared to the aerobic control group. Infusion supply of Klotho protein during IRI significantly reduced the level of phospho-IGF1R (p = 0.0436), PI3K (p = 0.0218) and phospho-AKT (p = 0.0020). Transcriptional activity of forkhead box protein O3 (FOXO3) was reduced (p = 0.0207) in hearts subjected to IRI, compared to aerobic control. Administration of Klotho decreased phosphorylation of FOXO3 (p = 0.0355), and enhanced activity of glutathione peroxidase (p = 0.0452) and superoxide dismutase (p = 0.0060) in IRI + Klotho group. The levels of reactive oxygen/nitrogen species (ROS/RNS) (p = 0.0480) and hydrogen peroxide (H2O2) (p = 0.0460), and heart injury (p = 0.0005) were significantly increased in hearts from the IRI group in comparison to the aerobic group. Klotho reduced NADPH oxidase 2 (NOX2) (p = 0.0390), ROS/RNS (p = 0.0435) and H2O2 (p = 0.0392) levels, and heart damage (p = 0.0286) in the hearts subjected to IRI. In conclusion, Klotho contributed to the protection of the heart against IRI and oxidative stress via inhibition of the IGF1R/PI3K/AKT pathway, thus can be recognized as a novel cardiopreventive/cardioprotective agent.

N-Feruloylserotonin inhibits lipopolysaccharide-induced inflammation via SIRT1-stimulated FOXO1 and NF-κB signaling pathways in RAW 264.7 cells.

Macrophages become activated by a variety of stimuli such as lipopolysaccharide (LPS) and participate in the process of immune responses. Activated macrophages produce various inflammatory mediators. In the present study, we investigated the anti-inflammatory mechanism of a serotonin derivative, N-feruloylserotonin, isolated from safflower seeds in RAW 264.7 macrophages. N-Feruloylserotonin treatment significantly attenuated these effects on LPS-induced reactive oxygen species, nitric oxide, and prostaglandin E2 production in RAW 264.7 macrophages. Furthermore, N-feruloylserotonin significantly decreased the abnormal expression of mitogen-activated protein kinase, such as phosphor (p)-c-Jun N-terminal kinase and p-extracellular-signal regulated kinase activation. Further research revealed that N-feruloylserotonin could stimulate sirtuin1 (SIRT1), then promote the forkhead box protein O1 (FOXO1), and suppress nuclear factor-kappa B (NF-kB) signaling pathways. The present study suggests that N-feruloylserotonin may be a new anti-inflammatory component and a promising candidate for anti-inflammatory therapeutic agents through the regulation of SIRT1-stimulated FOXO1 and NF-kB signaling pathways.

Identified senescence endotypes in aged cartilage are reflected in the blood metabolome

Heterogeneous accumulation of senescent cells expressing the senescence-associated secretory phenotype (SASP) affects tissue homeostasis which leads to diseases, such as osteoarthritis (OA). In this study, we set out to characterize heterogeneity of cellular senescence within aged articular cartilage and explored the presence of corresponding metabolic profiles in blood that could function as representative biomarkers. Hereto, we set out to perform cluster analyses, using a gene-set of 131 senescence genes (N = 57) in a previously established RNA sequencing dataset of aged articular cartilage and a generated metabolic dataset in overlapping blood samples. Using unsupervised hierarchical clustering and pathway analysis, we identified two robust cellular senescent endotypes. Endotype-1 was enriched for cell proliferating pathways, expressing forkhead box protein O4 (FOXO4), RB transcriptional corepressor like 2 (RBL2), and cyclin-dependent kinase inhibitor 1B (CDKN1B); the FOXO mediated cell cycle was identified as possible target for endotype-1 patients. Endotype-2 showed enriched inflammation-associated pathways, expressed by interleukin 6 (IL6), matrix metallopeptidase (MMP)1/3, and vascular endothelial growth factor (VEGF)C and SASP pathways were identified as possible targets for endotype-2 patients. Notably, plasma-based metabolic profiles in overlapping blood samples (N = 21) showed two corresponding metabolic clusters in blood. These non-invasive metabolic profiles could function as biomarkers for patient-tailored targeting of senescence in OA.

FGF2 promotes the chemotherapy resistance in colon cancer cells through activating PI3K/Akt signaling pathway

Abstract Background To investigate the role of fibroblast growth factor 2 (FGF2) in chemotherapy resistance of colon cancer. Methods An HCT116/5-fluorouracil (5-FU)–resistant cell line was established, and FGF2 levels were detected in a sensitive cell group (HCT116) and a resistant cell group (HCT1116-R) using different methods. Fibroblast growth factor 2 levels in the medium were determined by enzyme-linked immunoassay. The protein expressions of FGF2, fibroblast growth factor receptor 1 (FGFR1), and phospho-FGFR1 were assessed by Western blotting, and FGF2 mRNA levels were detected by quantitative real-time polymerase chain reaction. Fibroblast growth factor 2 recombinant protein was added to sensitive cells, and FGFR inhibitor AZD4547 was added to resistant cells, and the cell survival rate was determined using the cell counting kit-8 method and the protein expressions of PI3K (phosphatidylinositol 3 kinase), p-PI3K (phospho-PI3K), Akt (protein kinase B), p-Akt (phospho-Akt), mammalian target of rapamycin (mTOR), p-mTOR (phospho-mTOR), Bad (Bcl-xL/Bcl-2–associated death promoter), NF-κB (nuclear factor κB), GSK-3 (glycogen synthase kinase-3), FKHR (forkhead box protein O1), and PTEN (phosphatase and tensin homolog deleted on chromosome ten) were detected by Western blotting. Results Fibroblast growth factor 2 protein and mRNA expression levels in the HCT116-R group were significantly higher than those in the HCT116 group. Fibroblast growth factor 2 increased the survival rate of HCT116 cells; improved tolerance to 5-FU; upregulated p-PI3K, p-Akt, and p-mTOR; and downregulated Bad. The FGFR inhibitor AZD4547 decreased cell survival rate and tolerance to 5-FU; downregulated p-PI3K, p-Akt, and p-mTOR expression; and upregulated Bad. Conclusions Fibroblast growth factor 2 promotes chemotherapy tolerance in colon cancer cells by activating the Akt/mTOR and Akt/Bad signaling pathways downstream of PI3K.

The bcl6 corepressor mutation regulates the progression and transformation of myelodysplastic syndromes by repressing the autophagy flux

The occurrence of autophagy dysregulation is vital in the development of myelodysplastic syndrome and its transformation to acute myeloid leukemia. However, the mechanisms are largely unknown. Here, we have investigated the mechanism of the bcl6 corepressor mutation in myelodysplastic syndrome development and its transformation to acute myeloid leukemia. We identified a novel pathway involving histone deacetylase 6 and forkhead box protein O1, which leads to autophagy defects following the bcl6 corepressor mutation. And this further causes apoptosis and cell cycle arrest. The bcl6 corepressor-mutation-repressed autophagy resulted in the accumulation of damaged mitochondria, DNA, and reactive oxygen species in myelodysplastic syndrome cells, which could then lead to genomic instability and spontaneous mutation. Our results suggest that the bcl6 corepressor inactivating mutations exert pro-carcinogenic effects through survival strike, which is only an intermediate process. These findings provide mechanistic insights into the role of the bcl6 corepressor gene in myelodysplastic syndrome.