ABSTRACT
5-fluorouracil (5-FU) is an inexpensive treatment for colon cancer; however, its efficacy is limited by chemoresistance. This study investigates the combination therapy approach of 5-FU with Sitagliptin (Sita), a diabetic drug with potential cancer-modulating effects. The combination was evaluated in vitro and in silico, focusing on the effects of Sita and 5-FU on colon cancer cells. The results showed that the addition of Sita significantly decreased the IC50 of 5-FU compared to 5-Fu monotherapy. The study also found that Sita and 5-FU interact synergistically, with a combination index below 1. Sita successfully lowered the 5-FU dosage reduction index, decreasing the expression of MDR1 mRNA and p-AKT and NFκB2 subunits p100/p52 protein. Molecular docking analyses confirmed Sita's antagonistic action on MDR1 and thymidylate synthase proteins. The study concludes that sitagliptin can target MDR1, increase apoptosis, and significantly reduce the expression of p-AKT and NFκB2 cell-survival proteins. These effects sensitize colon cancer cells to 5-FU. Repurposing sitagliptin may enhance the anticancer effects of 5-FU at lower dosages.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B , Colonic Neoplasms , Drug Synergism , Fluorouracil , Proto-Oncogene Proteins c-akt , Sitagliptin Phosphate , Humans , Sitagliptin Phosphate/pharmacology , Fluorouracil/pharmacology , Colonic Neoplasms/metabolism , Colonic Neoplasms/drug therapy , Colonic Neoplasms/pathology , Proto-Oncogene Proteins c-akt/metabolism , ATP Binding Cassette Transporter, Subfamily B/metabolism , ATP Binding Cassette Transporter, Subfamily B/genetics , Down-Regulation/drug effects , Cell Line, Tumor , Molecular Docking SimulationABSTRACT
BACKGROUND: Nanoplastics (NPs) are emerging pollutants that pose risks to living organisms. Recent findings have unveiled the reproductive harm caused by polystyrene nanoparticles (PS-NPs) in female animals, yet the intricate mechanism remains incompletely understood. Under this research, we investigated whether sustained exposure to PS-NPs at certain concentrations in vivo can enter oocytes through the zona pellucida or through other routes that affect female reproduction. RESULTS: We show that PS-NPs disrupted ovarian functions and decreased oocyte quality, which may be a contributing factor to lower female fertility in mice. RNA sequencing of mouse ovaries illustrated that the PI3K-AKT signaling pathway emerged as the predominant environmental information processing pathway responding to PS-NPs. Western blotting results of ovaries in vivo and cells in vitro showed that PS-NPs deactivated PI3K-AKT signaling pathway by down-regulating the expression of PI3K and reducing AKT phosphorylation at the protein level, PI3K-AKT signaling pathway which was accompanied by the activation of autophagy and apoptosis and the disruption of steroidogenesis in granulosa cells. Since PS-NPs penetrate granulosa cells but not oocytes, we examined whether PS-NPs indirectly affect oocyte quality through granulosa cells using a granulosa cell-oocyte coculture system. Preincubation of granulosa cells with PS-NPs causes granulosa cell dysfunction, resulting in a decrease in the quality of the cocultured oocytes that can be reversed by the addition of 17ß-estradiol. CONCLUSIONS: This study provides findings on how PS-NPs impact ovarian function and include transcriptome sequencing analysis of ovarian tissue. The study demonstrates that PS-NPs impair oocyte quality by altering the functioning of ovarian granulosa cells. Therefore, it is necessary to focus on the research on the effects of PS-NPs on female reproduction and the related methods that may mitigate their toxicity.
Subject(s)
Granulosa Cells , Nanoparticles , Oocytes , Polystyrenes , Signal Transduction , Animals , Female , Mice , Apoptosis/drug effects , Autophagy/drug effects , Fertility/drug effects , Granulosa Cells/drug effects , Granulosa Cells/metabolism , Nanoparticles/toxicity , Oocytes/drug effects , Oocytes/metabolism , Ovary/drug effects , Ovary/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Polystyrenes/toxicity , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/drug effectsABSTRACT
Autophagy is closely related to the occurrence and development of human malignancies; however, the detailed mechanisms underlying autophagy in cervical cancer require further investigation. Previously, we found that the ectopic expression of NCAPH, a regulatory subunit of condensed protein complexes, significantly enhanced the proliferation of tumor cells; however, the underlying mechanisms were unclear. Here, we revealed that NCAPH is a novel autophagy-associated protein in cervical cancer that promotes cell proliferation by inhibiting autophagosome formation and reducing autophagy, with no effect on the cell cycle, apoptosis, or aging. Tripartite motif-containing protein 21 (TRIM21) is well known to be involved in inflammation, autoimmunity and cancer, mainly via its E3 ubiquitin ligase activity. Mass spectrometry and immunoprecipitation assays showed that TRIM21 interacted with NCAPH and decreased the protein stability of NCAPH via ubiquitination at the K11 lysine residue. Structural domain mutation analysis revealed that TRIM21 combined with NCAPH through its PRY/SPRY and CC domains and accelerated the degradation of NCAPH through the RING domain. Furthermore, TRIM21 promoted autophagosome formation and reduced cell proliferation by inhibiting NCAPH expression and the downstream AKT/mTOR pathway in cervical cancer cells. Immunohistochemical staining revealed that the protein expression of TRIM21 was negatively correlated with that of NCAPH and positively correlated with that of beclin-1 in cervical cancer tissues. Therefore, we provide evidence for the role of the TRIM21-NCAPH axis in cervical cancer autophagy and proliferation and the involvement of the AKT/mTOR signaling pathway in this process. These results deepen our understanding of the carcinogenesis of cervical cancer, broaden the understanding of the molecular mechanisms of TRIM21 and NCAPH, and provide guidance for individualized treatment of cervical cancer in the future.
Subject(s)
Autophagy , Cell Proliferation , Proto-Oncogene Proteins c-akt , Ribonucleoproteins , Signal Transduction , TOR Serine-Threonine Kinases , Ubiquitination , Uterine Cervical Neoplasms , Humans , Uterine Cervical Neoplasms/pathology , Uterine Cervical Neoplasms/metabolism , Uterine Cervical Neoplasms/genetics , Female , TOR Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Ribonucleoproteins/metabolism , Ribonucleoproteins/genetics , Cell Line, Tumor , Animals , HeLa Cells , Mice , Mice, NudeABSTRACT
BACKGROUND: High levels of lactate are positively associated with prognosis and mortality in pulmonary hypertension (PH). Lactate dehydrogenase A (LDHA) is a key enzyme for the production of lactate. This study is undertaken to investigate the role and molecular mechanisms of lactate and LDHA in PH. METHODS: Lactate levels were measured by a lactate assay kit. LDHA expression and localization were detected by western blot and Immunofluorescence. Proliferation and migration were determined by CCK8, western blot, EdU assay and scratch-wound assay. The right heart catheterization and right heart ultrasound were measured to evaluate cardiopulmonary function. RESULTS: In vitro, we found that lactate promoted proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) in an LDHA-dependent manner. In vivo, we found that LDHA knockdown reduced lactate overaccumulation in the lungs of mice exposed to hypoxia. Furthermore, LDHA knockdown ameliorated hypoxia-induced vascular remodeling and right ventricular dysfunction. In addition, the activation of Akt signaling by hypoxia was suppressed by LDHA knockdown both in vivo and in vitro. The overexpression of Akt reversed the inhibitory effect of LDHA knockdown on proliferation in PASMCs under hypoxia. Finally, LDHA inhibitor attenuated vascular remodeling and right ventricular dysfunction in Sugen/hypoxia mouse PH model, Monocrotaline (MCT)-induced rat PH model and chronic hypoxia-induced mouse PH model. CONCLUSIONS: Thus, LDHA-mediated lactate production promotes pulmonary vascular remodeling in PH by activating Akt signaling pathway, suggesting the potential role of LDHA in regulating the metabolic reprogramming and vascular remodeling in PH.
Subject(s)
Cell Proliferation , Hypertension, Pulmonary , L-Lactate Dehydrogenase , Lactate Dehydrogenase 5 , Lactic Acid , Mice, Inbred C57BL , Pulmonary Artery , Vascular Remodeling , Animals , Hypertension, Pulmonary/metabolism , Hypertension, Pulmonary/pathology , Hypertension, Pulmonary/physiopathology , Lactate Dehydrogenase 5/metabolism , Male , Lactic Acid/metabolism , L-Lactate Dehydrogenase/metabolism , Pulmonary Artery/pathology , Pulmonary Artery/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Cell Movement , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/pathology , Hypoxia/complications , Hypoxia/metabolism , Signal Transduction , Gene Knockdown Techniques , Mice , Cell Hypoxia , Rats, Sprague-Dawley , Rats , Humans , Lung/pathology , Lung/blood supplyABSTRACT
Peripheral nerve defect are common clinical problem caused by trauma or other diseases, often leading to the loss of sensory and motor function in patients. Autologous nerve transplantation has been the gold standard for repairing peripheral nerve defects, but its clinical application is limited due to insufficient donor tissue. In recent years, the application of tissue engineering methods to synthesize nerve conduits for treating peripheral nerve defect has become a current research focus. This study introduces a novel approach for treating peripheral nerve defects using a tissue-engineered PLCL/SF/NGF@TA-PPy-RGD conduit. The conduit was fabricated by combining electrospun PLCL/SF with an NGF-loaded conductive TA-PPy-RGD gel. The gel, synthesized from RGD-modified tannic acid (TA) and polypyrrole (PPy), provides growth anchor points for nerve cells. In vitro results showed that this hybrid conduit could enhance PC12 cell proliferation, migration, and reduce apoptosis under oxidative stress. Furthermore, the conduit activated the PI3K/AKT signalling pathway in PC12 cells. In a rat model of sciatic nerve defect, the PLCL/SF/NGF@TA-PPy-RGD conduit significantly improved motor function, gastrocnemius muscle function, and myelin sheath axon thickness, comparable to autologous nerve transplantation. It also promoted angiogenesis around the nerve defect. This study suggests that PLCL/SF/NGF@TA-PPy-RGD conduits provide a conducive environment for nerve regeneration, offering a new strategy for peripheral nerve defect treatment, this study provided theoretical basis and new strategies for the research and treatment of peripheral nerve defect.
Subject(s)
Hydrogels , Nerve Growth Factor , Nerve Regeneration , Oligopeptides , Phosphatidylinositol 3-Kinases , Proto-Oncogene Proteins c-akt , Sciatic Nerve , Signal Transduction , Animals , Nerve Regeneration/drug effects , Rats , Proto-Oncogene Proteins c-akt/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction/drug effects , PC12 Cells , Sciatic Nerve/drug effects , Sciatic Nerve/injuries , Oligopeptides/pharmacology , Oligopeptides/chemistry , Hydrogels/chemistry , Nerve Growth Factor/pharmacology , Nerve Growth Factor/metabolism , Rats, Sprague-Dawley , Male , Cell Proliferation/drug effects , Apoptosis/drug effects , Tissue Scaffolds/chemistry , Tissue Engineering/methods , Polymers/chemistrySubject(s)
Meningeal Neoplasms , Meningioma , Mutation , Proto-Oncogene Proteins c-akt , Tumor Necrosis Factor Receptor-Associated Peptides and Proteins , Humans , Meningioma/genetics , Meningioma/pathology , Meningeal Neoplasms/genetics , Meningeal Neoplasms/pathology , Female , Proto-Oncogene Proteins c-akt/genetics , Middle Aged , Male , Tumor Necrosis Factor Receptor-Associated Peptides and Proteins/genetics , Foramen Magnum/pathology , Adult , AgedABSTRACT
Background: Inhibiting ROS overproduction is considered a very effective strategy for the treatment of peripheral nerve injuries, and Se has a remarkable antioxidant effect; however, since the difference between the effective concentration of Se and the toxic dose is not large, we synthesized a nanomaterial that can release Se slowly so that it can be used more effectively. Methods: Se@SiO2 NPs were synthesized using a mixture of Cu2-x Se nanocrystals, and the mechanism of action of Se@SiO2 NPs was initially explored by performing sequencing, immunofluorescence staining and Western blotting of cellular experiments. The mechanism of action of Se@SiO2 NPs was further determined by performing behavioral assays after animal experiments and by sampling the material for histological staining, immunofluorescence staining, and ELISA. The effects, mechanisms and biocompatibility of Se@SiO2 NPs for peripheral nerve regeneration were determined. Results: Porous Se@SiO2 was successfully synthesized, had good particle properties, and could release Se slowly. CCK-8 experiments revealed that the optimal experimental doses were 100 µM H2O2 and 200 µg/mL Se@SiO2, and RNA-seq revealed that porous Se@SiO2 was associated with cell proliferation, apoptosis, and the PI3K/AKT pathway. WB showed that porous Se@SiO2 could increase the expression of cell proliferation antigens (PCNA and S100) and antiapoptotic proteins (Bcl-2), decrease the expression of proapoptotic proteins (Bax), and increase the expression of antioxidative stress proteins (Nrf2, HO-1, and SOD2). EdU cell proliferation and ROS fluorescence assays showed that porous Se@SiO2 promoted cell proliferation and reduced ROS levels. The therapeutic effect of LY294002 (a PI3K/AKT pathway inhibitor) was decreased significantly and its effect was lost when it was added simultaneously with porous Se@SiO2. Animal experiments revealed that the regenerated nerve fiber density, myelin thickness, axon area, gastrocnemius muscle wet-to-weight ratio, myofiber area, sciatic nerve function index (SFI), CMAP, apoptotic cell ratio, and levels of antioxidative stress proteins and anti-inflammatory factors were increased following the administration of porous Se@SiO2. The levels of oxidative stress proteins and anti-inflammatory factors were significantly greater in the Se@SiO2 group than in the PNI group, and the effect of LY294002 was decreased significantly and was lost when it was added simultaneously with porous Se@SiO2. Conclusion: Se@SiO2 NPs are promising, economical and effective Se-releasing nanomaterials that can effectively reduce ROS production, inhibit apoptosis and promote cell proliferation after nerve injury via the PI3K/AKT pathway, ultimately accelerating nerve regeneration. These findings could be used to design new, promising drugs for the treatment of peripheral nerve injury.
Subject(s)
Cell Proliferation , Nerve Regeneration , Peripheral Nerve Injuries , Phosphatidylinositol 3-Kinases , Proto-Oncogene Proteins c-akt , Selenium , Signal Transduction , Silicon Dioxide , Animals , Selenium/chemistry , Selenium/pharmacology , Silicon Dioxide/chemistry , Silicon Dioxide/pharmacology , Peripheral Nerve Injuries/drug therapy , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Nerve Regeneration/drug effects , Cell Proliferation/drug effects , Rats , Apoptosis/drug effects , Antioxidants/pharmacology , Antioxidants/chemistry , Nanoparticles/chemistry , Male , Delayed-Action Preparations/pharmacology , Delayed-Action Preparations/chemistry , Rats, Sprague-Dawley , Oxidative Stress/drug effects , Sciatic Nerve/drug effects , Sciatic Nerve/injuries , Schwann Cells/drug effects , Schwann Cells/metabolismABSTRACT
The study investigated the protective effect and mechanism of 2-phenylethyl-beta-glucopyranoside(Phe) from Huaizhong No.1 Rehmannia glutinosa on hypoxic pulmonary hypertension(PH), aiming to provide a theoretical basis for clinical treatment of PAH. Male C57BL/6N mice were randomly divided into normal group, model group, positive drug(bosentan, 100 mg·kg~(-1)) group, and low-and high-dose Phe groups(20 and 40 mg·kg~(-1)). Except for the normal group, all other groups were continuously subjected to model induction in a 10% hypoxic environment for 5 weeks, with oral administration for 14 days starting from the 3rd week. The cardiopulmonary function, right ventricular pressure, cough and asthma index, lung injury, cell apoptosis, oxidative stress-related indicators, immune cells, and phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR)/hypoxic inducible factor 1α(HIF-1α) pathway-related proteins or mRNA levels were examined. Furthermore, hypoxia-induced pulmonary arterial smooth muscle cell(PASMC) were used to further explore the mechanism of Phe intervention in PH combined with PI3K ago-nist(740Y-P). The results showed that Phe significantly improved the cardiopulmonary function of mice with PH, decreased right ventricular pressure, cough and asthma index, and lung injury, reduced cell apoptosis, oxidative stress-related indicators, and nuclear levels of phosphorylated Akt(p-Akt) and phosphorylated mTOR(p-mTOR), inhibited the expression levels of HIF-1α and PI3K mRNA and proteins, and maintained the immune cell homeostasis in mice. Further mechanistic studies revealed that Phe significantly reduced the viability and migration ability of hypoxia-induced PASMC, decreased the expression of HIF-1α and PI3K proteins and nuc-lear levels of p-Akt and p-mTOR, and this effect was blocked by 740Y-P. Therefore, it is inferred that Phe may exert anti-PH effects by alleviating the imbalance of oxidative stress and apoptosis in lung tissues and regulating immune levels, and its mechanism may be related to the regulation of the PI3K/Akt/mTOR/HIF-1α pathway. This study is expected to provide drug references and research ideas for the treatment of PH.
Subject(s)
Glucosides , Hypertension, Pulmonary , Hypoxia-Inducible Factor 1, alpha Subunit , Hypoxia , Mice, Inbred C57BL , Phosphatidylinositol 3-Kinases , Proto-Oncogene Proteins c-akt , Rehmannia , TOR Serine-Threonine Kinases , Animals , Male , TOR Serine-Threonine Kinases/metabolism , TOR Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-akt/genetics , Mice , Hypertension, Pulmonary/drug therapy , Hypertension, Pulmonary/physiopathology , Hypertension, Pulmonary/metabolism , Hypertension, Pulmonary/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Rehmannia/chemistry , Phosphatidylinositol 3-Kinases/metabolism , Phosphatidylinositol 3-Kinases/genetics , Glucosides/pharmacology , Hypoxia/drug therapy , Hypoxia/physiopathology , Hypoxia/metabolism , Signal Transduction/drug effects , Humans , Drugs, Chinese Herbal/pharmacology , Drugs, Chinese Herbal/administration & dosage , Drugs, Chinese Herbal/chemistry , Apoptosis/drug effectsABSTRACT
To investigate the mechanism by which Peitu Yifei Granules inhibit idiopathic pulmonary fibrosis(IPF) in rats, fifty specific-pathogen-free(SPF) grade male Wistar rats were randomly divided into blank group and modeling group. IPF was induced in the modeling group rats by tracheal infusion of 5 mg·kg~(-1) bleomycin(BLM) and then randomly divided into model group, pirfenidone group, and high-dose, medium-dose, and low-dose groups treated with Peitu Yifei Granules. After 24 hours of modeling, the treatment groups received intragastric administration of either Peitu Yifei Granules or pirfenidone as a positive control drug; meanwhile, the model group received an equal volume of normal saline. After 21 days of treatment administration, lung tissue samples were collected for analysis. Pathological changes in lung tissues were assessed using hematoxylin-eosin(HE) staining and Masson's trichrome staining. The expression levels of protein kinase B(Akt), mammalian target of rapamycin(mTOR), their phosphorylated forms, and sequestosome 1(p62) were determined through Western blot(WB). Fluorescent quantitative real-time polymerase chain reaction(RT-qPCR) was used to measure messenger ribonucleic acid(mRNA) expression levels of Beclin-1, microtubule-associated proteins 1A/1B light chain 3B(LC3B), and p62. Immunohistochemistry was performed to assess protein expression levels of Beclin-1 and LC3B in lung tissue samples. RESULTS:: demonstrated that lung tissue structure appeared normal without significant collagen deposition in the blank group rats. In contrast, rats from the model group exhibited thickened alveolar septa along with evident inflammatory changes and collagen deposition. Compared to the model group rats, those treated with Peitu Yifei Granules or pirfenidone showed significantly improved lung tissue structure with reduced inflammation and collagen deposition observed histologically. Furthermore, compared with those of the blank group, the expressions of p62 and its mRNA, p-Akt and p-mTOR protein in lung tissues of the model group were significantly increased, while Beclin-1, LC3B and their mRNA levels were significantly decreased. Compared with those of the model group, the expressions of p62 and its mRNA, p-Akt and p-mTOR in lung tissues of the pirfenidone group and Peitu Yifei Granules high-dose and medium-dose groups were significantly decreased, while Beclin-1, LC3B and their mRNA expressions were significantly increased. The above results indicate that Peitu Yifei Granules can improve autophagy levels in lung tissues by inhibiting the phosphoinositide 3-kinase(PI3K)/Akt/mTOR signaling pathway and delay the development of IPF disease.
Subject(s)
Autophagy , Drugs, Chinese Herbal , Idiopathic Pulmonary Fibrosis , Phosphatidylinositol 3-Kinases , Proto-Oncogene Proteins c-akt , Rats, Wistar , Signal Transduction , TOR Serine-Threonine Kinases , Animals , TOR Serine-Threonine Kinases/metabolism , TOR Serine-Threonine Kinases/genetics , Male , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-akt/genetics , Rats , Drugs, Chinese Herbal/pharmacology , Drugs, Chinese Herbal/administration & dosage , Autophagy/drug effects , Signal Transduction/drug effects , Idiopathic Pulmonary Fibrosis/drug therapy , Idiopathic Pulmonary Fibrosis/metabolism , Idiopathic Pulmonary Fibrosis/genetics , Phosphatidylinositol 3-Kinases/metabolism , Phosphatidylinositol 3-Kinases/genetics , Lung/drug effects , Lung/metabolism , Lung/pathology , HumansABSTRACT
BACKGROUND: The global prevalence of autoimmune hepatitis (AIH) is increasing due in part to the lack of effective pharmacotherapies. Growing evidence suggests that fibroblast growth factor 4 (FGF4) is crucial for diverse aspects of liver pathophysiology. However, its role in AIH remains unknown. Therefore, we investigated whether FGF4 can regulate M1 macrophage and thereby help treat liver inflammation in AIH. METHODS: We obtained transcriptome-sequencing and clinical data for patients with AIH. Mice were injected with concanavalin A to induce experimental autoimmune hepatitis (EAH). The mechanism of action of FGF4 was examined using macrophage cell lines and bone marrow-derived macrophages. RESULTS: We observed higher expression of markers associated with M1 and M2 macrophages in patients with AIH than that in individuals without AIH. EAH mice showed greater M1-macrophage polarization than control mice. The expression of M1-macrophage markers correlated positively with FGF4 expression. The loss of hepatic Fgf4 aggravated hepatic inflammation by increasing the abundance of M1 macrophages. In contrast, the pharmacological administration of FGF4 mitigated hepatic inflammation by reducing M1-macrophage levels. The efficacy of FGF4 treatment was compromised following the in vivo clearance of macrophage populations. Mechanistically, FGF4 treatment activated the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT)-signal pathway in macrophages, which led to reduced M1 macrophages and hepatic inflammation. CONCLUSION: We identified FGF4 as a novel M1/M2 macrophage-phenotype regulator that acts through the PI3K-AKT-signaling pathway, suggesting that FGF4 may represent a novel target for treating inflammation in patients with AIH.
Subject(s)
Cell Polarity , Fibroblast Growth Factor 4 , Hepatitis, Autoimmune , Inflammation , Macrophages , Mice, Inbred C57BL , Animals , Female , Humans , Male , Mice , Cell Polarity/drug effects , Disease Models, Animal , Fibroblast Growth Factor 4/metabolism , Hepatitis, Autoimmune/pathology , Hepatitis, Autoimmune/metabolism , Inflammation/pathology , Liver/pathology , Liver/metabolism , Liver/drug effects , Macrophage Activation/drug effects , Macrophages/metabolism , Macrophages/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/drug effectsABSTRACT
Epithelial-mesenchymal transition (EMT) is a cellular process in embryonic development, wound healing, organ fibrosis, and cancer metastasis. Previously, we and others have reported that proinflammatory cytokine interleukin-1ß (IL-1ß) induces EMT. However, the exact mechanisms, especially the signal transduction pathways, underlying IL-1ß-mediated EMT are not yet completely understood. Here, we found that IL-1ß stimulation leads to the partial EMT-like phenotype in human lung epithelial A549 cells, including the gain of mesenchymal marker (vimentin) and high migratory potential, without the complete loss of epithelial marker (E-cadherin). IL-1ß-mediated partial EMT induction was repressed by PI3K inhibitor LY294002, indicating that the PI3K/AKT pathway plays a significant role in the induction. In addition, ERK1/2 inhibitor FR180204 markedly inhibited the IL-1ß-mediated partial EMT induction, demonstrating that the MEK/ERK pathway was also involved in the induction. Furthermore, we found that the activation of the PI3K/AKT and MEK/ERK pathways occurred downstream of the epidermal growth factor receptor (EGFR) pathway and the IL-1 receptor (IL-1R) pathway, respectively. Our findings suggest that the PI3K/AKT and MEK/ERK pathways coordinately promote the IL-1ß-mediated partial EMT induction. The inhibition of not one but both pathways is expected yield clinical benefits by preventing partial EMT-related disorders such as organ fibrosis and cancer metastasis.
Subject(s)
Epithelial-Mesenchymal Transition , Interleukin-1beta , MAP Kinase Signaling System , Phosphatidylinositol 3-Kinases , Proto-Oncogene Proteins c-akt , Humans , Interleukin-1beta/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Phosphatidylinositol 3-Kinases/metabolism , A549 Cells , ErbB Receptors/metabolismABSTRACT
BACKGROUND: Patients with endometriosis suffer with chronic pelvic pain and infertility, and from the lack of pharmacologic therapies that consistently halt disease progression. Differences in the endometrium of patients with endometriosis vs. unaffected controls are well-documented. Specifically, shed endometrial tissues (delivered to the pelvic cavity via retrograde menstruation) reveal that a subset of stromal cells exhibiting pro-inflammatory, pro-fibrotic, and pro-senescence-like phenotypes is enhanced in endometriosis patients compared to controls. Additionally, cultured biopsy-derived endometrial stromal cells from endometriosis patients exhibit impaired decidualization, a defined differentiation process required for human embryo implantation and pregnancy. Quercetin, a senolytic agent, shows therapeutic potential for pulmonary fibrosis, a disorder attributed to senescent pulmonary fibroblasts. In rodent models of endometriosis, quercetin shows promise, and quercetin improves decidualization in vitro. However, the exact mechanisms are not completely understood. Therefore, we investigated the effects of quercetin on menstrual effluent-derived endometrial stromal cells from endometriosis patients and unaffected controls to define the signaling pathways underlying quercetin's effects on endometrial stromal cells. METHODS: Menstrual effluent-derived endometrial stromal cells were collected and cultured from unaffected controls and endometriosis patients and then, low passage cells were treated with quercetin (25 µM) under basal or standard decidualization conditions. Decidualization responses were analyzed by measuring the production of IGFBP1 and PRL. Also, the effects of quercetin on intracellular cAMP levels and cellular oxidative stress responses were measured. Phosphokinase arrays, western blotting, and flow cytometry methods were performed to define the effects of quercetin on various signaling pathways and the potential mechanistic roles of quercetin. RESULTS: Quercetin significantly promotes decidualization of control- and endometriosis-endometrial stromal cells. Quercetin substantially reduces the phosphorylation of multiple signaling molecules in the AKT and ERK1/2 pathways, while enhancing the phosphorylation of p53 and total p53 levels. Furthermore, p53 inhibition blocks decidualization while p53 activation promotes decidualization. Finally, we provide evidence that quercetin increases apoptosis of endometrial stromal cells with a senescent-like phenotype. CONCLUSIONS: These data provide insight into the mechanisms of action of quercetin on endometrial stromal cells and warrant future clinical trials to test quercetin and other senolytics for treating endometriosis.
Subject(s)
Cellular Senescence , Endometriosis , Proto-Oncogene Proteins c-akt , Quercetin , Stromal Cells , Tumor Suppressor Protein p53 , Quercetin/pharmacology , Female , Humans , Endometriosis/metabolism , Endometriosis/pathology , Endometriosis/drug therapy , Proto-Oncogene Proteins c-akt/metabolism , Adult , Stromal Cells/drug effects , Stromal Cells/metabolism , Cellular Senescence/drug effects , Tumor Suppressor Protein p53/metabolism , Endometrium/drug effects , Endometrium/metabolism , Endometrium/pathology , Decidua/drug effects , Decidua/metabolism , Signal Transduction/drug effects , MAP Kinase Signaling System/drug effects , Cells, CulturedABSTRACT
Malva parviflora has shown anti-inflammatory, antihypertensive, antihyperlipidemic, and hypoglycemic effects. This study is aimed to evaluate the anti-adipogenic effect of M. parviflora on 3T3-L1 adipocytes. Fibroblast differentiation was induced either in the absence or presence of M. parviflora fractions (F3, F4, F7, F12, F13, F17, F18 and F19) for 4 days; F17 and 18 were the most effective fractions in reducing intracellular lipid accumulation (by 25.6% and 23.1%, respectively). EC50 of F17 and F18 (14 µg/mL and 17 µg/mL, respectively) were used to evaluate their anti adipogenic effect. After 10 days of inducing differentiation in the absence or presence of the extracts at the EC50 of F17 and F18, lipid accumulation, the concentration of interleukin 6 (IL-6) were measured in the culture medium; the presence of PPAR-γ, AKT, and p-AKT was also determined. In differentiated adipocytes (C2), F17 maintained intracellular lipid concentration at levels comparable to metformin, while decreasing PPAR-γ and increasing p-AKT presence; it also prevented IL-6 expression. F17 consists of alanine, valine, phenylalanine, and proline. On the other hand, F18 reduced intracellular lipid concentrations, prevented the increase of PPAR-γ and p-AKT, and maintained IL-6 expression at similar levels as metformin. F18 is mainly constituted by alanine, valine, proline, and sucrose. In conclusion, M. parviflora fractions (F17 and F18) control the process of adipogenesis, lipogenesis, and cellular dysfunction.
Subject(s)
3T3-L1 Cells , Adipocytes , Adipogenesis , PPAR gamma , Plant Extracts , Animals , Mice , Adipocytes/drug effects , Adipocytes/metabolism , Adipocytes/cytology , Adipogenesis/drug effects , Plant Extracts/pharmacology , PPAR gamma/metabolism , Interleukin-6/metabolism , Cell Differentiation/drug effects , Lipid Metabolism/drug effects , Proto-Oncogene Proteins c-akt/metabolismABSTRACT
Circular RNA (circRNA) is thought to mediate the occurrence and development of human cancer and usually acts as a tiny RNA (miRNA) sponge to regulate downstream gene expression. However, it is not clear whether and how circACVR2A (hsa_circ_0001073) is involved in the progression of HCC. The purpose of this study is to clarify the potential role and molecular mechanism of circACVR2A in regulating the progression of hepatocellular carcinoma cells (HCC). The abundance of related proteins in circACVR2A, microRNA (miR511-5p) and PI3K-Akt signaling pathway was determined by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) or Western blotting. Cell viability, invasion and apoptosis were analyzed by CCK-8, Transwell analysis and Tunel staining, respectively. The interaction between circACVR2A and microRNA was evaluated by double luciferase reporter gene assay. The results showed that circACVR2A was highly expressed in hepatocellular carcinoma cell lines. Our in vivo and in vitro data showed that circACVR2A promoted the proliferation, migration and invasion of HCC. In terms of mechanism, we found that circACVR2A can directly interact with miR511-5p and act as a miRNA sponge to regulate the expression of related proteins in PI3K-Akt signaling pathway.In HCC, circACVR2A can mediate miR-511-5p/mRNA network to activate PI3K signal pathway. This shows that the molecular regulatory network with circACVR2A as the core is a new potential target for diagnosis and treatment of hepatocellular carcinoma.
Subject(s)
Carcinoma, Hepatocellular , Cell Movement , Cell Proliferation , Gene Expression Regulation, Neoplastic , Liver Neoplasms , MicroRNAs , Phosphatidylinositol 3-Kinases , Proto-Oncogene Proteins c-akt , RNA, Circular , Signal Transduction , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/metabolism , MicroRNAs/genetics , Humans , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Liver Neoplasms/metabolism , RNA, Circular/genetics , Proto-Oncogene Proteins c-akt/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Cell Movement/genetics , Animals , Cell Line, Tumor , Mice , Apoptosis/genetics , Disease Progression , MaleABSTRACT
Catheter ablation (CA) is an essential method for the interventional treatment of atrial fibrillation (AF), and it is very important to reduce long-term recurrence after CA. The mechanism of recurrence after CA is still unclear. We established a long-term model of beagle canines after circumferential pulmonary vein ablation (CPVA). The transcriptome and proteome were obtained using high-throughput sequencing and TMT-tagged LC-MS/LC analysis, respectively. Differentially expressed genes and proteins were screened and enriched, and the effect of fibrosis was found and verified in tissues. A downregulated protein, neuropeptide Y (NPY), was selected for validation and the results suggest that NPY may play a role in the long-term reinduction of AF after CPVA. Then, the molecular mechanism of NPY was further investigated. The results showed that the atrial effective refractory period (AERP) was shortened and fibrosis was increased after CPVA. Atrial myocyte apoptosis was alleviated by NPY intervention, and Akt activation was inhibited in cardiac fibroblasts. These results suggest that long-term suppression of NPY after CPVA may lead to induction of AF through promoting cardiomyocyte apoptosis and activating the Akt pathway in cardiac fibroblasts, which may make AF more likely to reinduce.
Subject(s)
Apoptosis , Atrial Fibrillation , Catheter Ablation , Myocardium , Neuropeptide Y , Pulmonary Veins , Animals , Dogs , Apoptosis/drug effects , Atrial Fibrillation/metabolism , Atrial Fibrillation/surgery , Atrial Fibrillation/pathology , Catheter Ablation/methods , Disease Models, Animal , Fibrosis , Heart Atria/metabolism , Heart Atria/pathology , Multiomics , Myocardium/metabolism , Myocardium/pathology , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/pathology , Neuropeptide Y/metabolism , Proteome/metabolism , Proteomics/methods , Proto-Oncogene Proteins c-akt/metabolism , Pulmonary Veins/metabolism , Pulmonary Veins/surgery , TranscriptomeABSTRACT
Background: Ulcerative colitis (UC) is an intestinal inflammatory disease that is strongly associated with mitochondrial damage and dysfunction as well as mitophagy and lacks of satisfactory treatments. Hair follicle mesenchymal stem cell (HF-MSC)-derived exosomes owe benefit effectiveness on inflammatory therapies. Hypoxia-preconditioned HF-MSCs exhibit enhanced proliferation and migration abilities, and their exosomes exert stronger effects than normal exosomes. However, the therapeutic function of Hy-Exos in UC is unknown. Methods: The inflammation model was established with LPS-treated MODE-K cells, and the mouse UC model was established by dextran sulfate sodium (DSS) administration. The therapeutic effects of HF-MSC-derived exosomes (Exos) and hypoxia-preconditioned HF-MSC-derived exosomes (Hy-Exos) were compared in vitro and in vivo. Immunofluorescence staining and western blotting were used to explore the effects of Hy-Exos on mitochondrial function, mitochondrial fission and fusion and mitophagy. MiRNA sequencing analysis was applied to investigate the differences in components between Exos and Hy-Exos. Results: Hy-Exos had a better therapeutic effect on LPS-treated MODE-K cells and DSS-induced UC mice. Hy-Exos promoted colonic tight junction proteins expression, suppressed the oxidative stress response, and reduced UC-related inflammatory injury. Hy-Exos may exert these effects via miR-214-3p-mediated inhibition of the PI3K/AKT/mTOR signaling pathway, maintenance of mitochondrial dynamic stability, alleviation of mitochondrial dysfunction and enhancement of mitophagy. Conclusion: This study revealed a vital role for Hy-Exos in suppressing inflammatory progression in UC and suggested that miR-214-3p is a potential critical target for Hy-Exos in alleviating UC.
Subject(s)
Colitis, Ulcerative , Disease Models, Animal , Exosomes , Hair Follicle , Mesenchymal Stem Cells , Mitophagy , Phosphatidylinositol 3-Kinases , Proto-Oncogene Proteins c-akt , Signal Transduction , TOR Serine-Threonine Kinases , Animals , Colitis, Ulcerative/metabolism , Colitis, Ulcerative/therapy , Colitis, Ulcerative/pathology , Mesenchymal Stem Cells/metabolism , Exosomes/metabolism , Mice , Proto-Oncogene Proteins c-akt/metabolism , TOR Serine-Threonine Kinases/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Hair Follicle/metabolism , Dextran Sulfate , Male , Mitochondria/metabolism , Mice, Inbred C57BL , MicroRNAs/metabolism , MicroRNAs/genetics , HumansABSTRACT
Globally, the prevalence of breast cancer (BC) is increasing at an alarming level, despite early detection and technological improvements. Alkaloids are diverse chemical groups, and many within this class have been reported as potential anticancer compounds. Chabamide F (F) and chabamide G (G) are two dimeric amide alkaloids found in a traditional medicinal plant, Piper chaba, and possess significant cytotoxic effects. However, their scientific rationalization in BC remains unknown. Here, we aimed to investigate their potential and molecular mechanisms for BC through in silico approaches. From network pharmacology, we identified 64 BC-related genes as targets. GO and KEGG studies showed that they were involved in various biological processes and mostly expressed in BC-related pathways such as RAS, PI3K-AKT, estrogen, MAPK, and FoxO pathways. However, PPI analysis revealed SRC and AKT1 as hub genes, which play key roles in BC tumorigenesis and metastasis. Molecular docking revealed the strong binding affinity of F (- 10.7 kcal/mol) and G (- 9.4 and - 11.7 kcal/mol) for SRC and AKT1, respectively, as well as the acquisition of vital residues to inhibit them. Their long-term stability was evaluated using 200 ns molecular dynamics simulation. The RMSD, RMSF, Rg, and SASA analyses showed that the G-SRC and G-AKT1 complexes were excellently stable compared to the control, dasatinib, and capivasertib, respectively. Additionally, the PCA and DCCM analyses revealed a significant reduction in the residual correlation and motions. By contrast, the stability of the F-SRC complex was greater than that of the control, whereas it was moderately stable in complex with AKT1. The MMPBSA analysis demonstrated higher binding energies for both compounds than the controls. In particular, the binding energy of G for SRC and AKT1 was - 120.671 ± 16.997 and - 130.437 ± 19.111 kJ/mol, respectively, which was approximately twice as high as the control molecules. Van der Waal and polar solvation energies significantly contributed to this energy. Furthermore, both of them exhibited significant interactions with the binding site residues of both proteins. In summary, this study indicates that these two molecules could be a potential ATP-competitive inhibitor of SRC and an allosteric inhibitor of AKT1.
Subject(s)
Breast Neoplasms , Computational Biology , Molecular Docking Simulation , Proto-Oncogene Proteins c-akt , Breast Neoplasms/genetics , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Humans , Female , Computational Biology/methods , Proto-Oncogene Proteins c-akt/metabolism , Molecular Dynamics Simulation , src-Family Kinases/metabolism , src-Family Kinases/geneticsABSTRACT
Depressive pseudodementia (DPD) is a debilitating cognitive dysfunction that accompanies major and/or frequent depressive attacks. DPD has gained significant research attention owing to its negative effects on the patients' quality of life and productivity. This study tested the procognitive potential of Flibanserin (FBN), the serotonin (5HT) receptor modulator, against propranolol (PRP), as ß/5HT1A receptors blocker. Serving this purpose, female Wistar Albino rats were subjected to chronic unpredictable stress (CUS) and subsequently treated with FBN only (3 mg/kg/day, p.o), PRP only (10 mg/kg/day, p.o), or PRP followed by FBN, using the same doses. FBN ameliorated the behavioral/cognitive alterations and calmed the hypothalamic-pituitary-adrenal (HPA) axis storm by reducing the levels of stress-related hormones, viz, corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), corticosterone (CORT) parallel to epinephrine (EPI) hyperstimulation. The maladaptive inflammatory response, comprising of interleukin (IL)-1ß/6, and tumor necrosis factor (TNF)-α, was consequently blunted. This was contemporaneous to the partial restoration of the protein kinase-B (AKT)/glycogen synthase kinase (GSK)3ß/signal transducer and activator of transcription (STAT)-3 survival trajectory and the reinstatement of the levels of brain derived neurotrophic factor (BDNF). Microscopically, FBN repaired the hippocampal architecture and lessened CD68/GFAP immunoreactivity. Pre-administration of PRP partially abolished FBN effect along the estimated parameters, except for 5HT2A receptor expression and epinephrine level, to prove 5HT1A receptor as a fulcrum initiator of the investigated pathway, while its sole administration worsened the underlying condition. Ultimately, these findings highlight the immense procognitive potential of FBN, offering a new paradigm for halting DPD advancement via synchronizing adrenergic/serotonergic circuitry.
Subject(s)
Benzimidazoles , Brain-Derived Neurotrophic Factor , Hypothalamo-Hypophyseal System , Pituitary-Adrenal System , Proto-Oncogene Proteins c-akt , Rats, Wistar , Animals , Female , Rats , Behavior, Animal/drug effects , Brain-Derived Neurotrophic Factor/metabolism , Depression/drug therapy , Depression/metabolism , Disease Models, Animal , Hypothalamo-Hypophyseal System/drug effects , Hypothalamo-Hypophyseal System/metabolism , Inflammation/drug therapy , Inflammation/pathology , Inflammation/metabolism , Pituitary-Adrenal System/drug effects , Pituitary-Adrenal System/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Serotonin/metabolism , Signal Transduction/drug effects , Stress, Psychological/drug therapy , Stress, Psychological/complications , Benzimidazoles/pharmacology , Benzimidazoles/therapeutic useABSTRACT
The signaling complex around voltage-gated sodium (Nav) channels includes accessory proteins and kinases crucial for regulating neuronal firing. Previous studies showed that one such kinase, WEE1-critical to the cell cycle-selectively modulates Nav1.2 channel activity through the accessory protein fibroblast growth factor 14 (FGF14). Here, we tested whether WEE1 exhibits crosstalk with the AKT/GSK3 kinase pathway for coordinated regulation of FGF14/Nav1.2 channel complex assembly and function. Using the in-cell split luciferase complementation assay (LCA), we found that the WEE1 inhibitor II and GSK3 inhibitor XIII reduce the FGF14/Nav1.2 complex formation, while the AKT inhibitor triciribine increases it. However, combining WEE1 inhibitor II with either one of the other two inhibitors abolished its effect on the FGF14/Nav1.2 complex formation. Whole-cell voltage-clamp recordings of sodium currents (INa) in HEK293 cells co-expressing Nav1.2 channels and FGF14-GFP showed that WEE1 inhibitor II significantly suppresses peak INa density, both alone and in the presence of triciribine or GSK3 inhibitor XIII, despite the latter inhibitor's opposite effects on INa. Additionally, WEE1 inhibitor II slowed the tau of fast inactivation and caused depolarizing shifts in the voltage dependence of activation and inactivation. These phenotypes either prevailed or were additive when combined with triciribine but were outcompeted when both WEE1 inhibitor II and GSK3 inhibitor XIII were present. Concerted regulation by WEE1 inhibitor II, triciribine, and GSK3 inhibitor XIII was also observed in long-term inactivation and use dependency of Nav1.2 currents. Overall, these findings suggest a complex role for WEE1 kinase-in concert with the AKT/GSK3 pathway-in regulating the Nav1.2 channelosome.
Subject(s)
Cell Cycle Proteins , Glycogen Synthase Kinase 3 , NAV1.2 Voltage-Gated Sodium Channel , Protein-Tyrosine Kinases , Proto-Oncogene Proteins c-akt , Humans , HEK293 Cells , Proto-Oncogene Proteins c-akt/metabolism , Cell Cycle Proteins/metabolism , NAV1.2 Voltage-Gated Sodium Channel/metabolism , NAV1.2 Voltage-Gated Sodium Channel/genetics , Protein-Tyrosine Kinases/metabolism , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3/antagonists & inhibitors , Fibroblast Growth Factors/metabolism , Signal Transduction/drug effectsABSTRACT
From birth to adulthood, the mammalian heart grows primarily through increasing cardiomyocyte (CM) size, which is known as maturational hypertrophic growth. The Hippo-YAP signaling pathway is well known for regulating heart development and regeneration, but its roles in CM maturational hypertrophy have not been clearly addressed. Vestigial-like 4 (VGLL4) is a crucial component of the Hippo-YAP pathway, and it functions as a suppressor of YAP/TAZ, the terminal transcriptional effectors of this signaling pathway. To develop an in vitro model for studying CM maturational hypertrophy, we compared the biological effects of T3 (triiodothyronine), Dex (dexamethasone), and T3/Dex in cultured neonatal rat ventricular myocytes (NRVMs). The T3/Dex combination treatment stimulated greater maturational hypertrophy than either the T3 or Dex single treatment. Using T3/Dex treatment of NRVMs as an in vitro model, we found that activation of VGLL4 suppressed CM maturational hypertrophy. In the postnatal heart, activation of VGLL4 suppressed heart growth, impaired heart function, and decreased CM size. On the molecular level, activation of VGLL4 inhibited the PI3K-AKT pathway, and disrupting VGLL4 and TEAD interaction abolished this inhibition. In conclusion, our data suggest that VGLL4 suppresses CM maturational hypertrophy by inhibiting the YAP/TAZ-TEAD complex and its downstream activation of the PI3K-AKT pathway.