Akt Phosphorylation Research Articles

Protein kinase D2 modulates hepatic insulin sensitivity in male mice

ObjectivesProtein kinase D (PKD) family is emerging as relevant regulator of metabolic homeostasis. However, the precise role of PKD2 in modulating hepatic insulin signaling has not been fully elucidated and it is the aim of this study. MethodsPKD inhibition was analyzed for insulin signaling in mouse and human hepatocytes. PKD2 was overexpressed in Huh7 hepatocytes and mouse liver, and insulin responses were evaluated. Mice with hepatocyte-specific PKD2 depletion (PKD2ΔHep) and PKD2fl/fl mice were fed a chow (CHD) or high fat diet (HFD) and glucose homeostasis and lipid metabolism were investigated. ResultsPKD2 silencing enhanced insulin signaling in hepatocytes, an effect also found in primary hepatocytes from PKD2ΔHep mice. Conversely, a constitutively active PKD2 mutant reduced insulin-stimulated AKT phosphorylation. A more in-depth analysis revealed reduced IRS1 serine phosphorylation under basal conditions and increased IRS1 tyrosine phosphorylation in PKD2ΔHep primary hepatocytes upon insulin stimulation and, importantly PKD co-immunoprecipitates with IRS1. In vivo constitutively active PKD2 overexpression resulted in a moderate impairment of glucose homeostasis and reduced insulin signaling in the liver. On the contrary, HFD-fed PKD2ΔHep male mice displayed improved glucose and pyruvate tolerance, as well as higher peripheral insulin tolerance and enhanced hepatic insulin signaling compared to control PKD2fl/fl mice. Despite of a remodeling of hepatic lipid metabolism in HFD-fed PKD2ΔHep mice, similar steatosis grade was found in both genotypes. ConclusionsResults herein have unveiled an unknown role of PKD2 in the control of insulin signaling in the liver at the level of IRS1 and point PKD2 as a therapeutic target for hepatic insulin resistance.

PTGES is involved in myofibroblast differentiation via HIF-1α-dependent glycolysis pathway.

Lung cancer is the leading cause of cancer-related deaths worldwide. Patients with lung cancer usually exhibit poor prognoses and low 5-year survival rates. Idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD) are both chronic lung dysfunctions resulting in lung fibrosis and increased risk of lung cancer. Myofibroblasts contribute to the progression of asthma, COPD and IPF, leading to fibrosis in the airway and lungs. A growing body of evidence demonstrates that metabolic reprogramming is a major hallmark of fibrosis, being important in the progression of fibrosis. Using gene expression microarray, we identified and validated that the lipid metabolic pathway was upregulated in lung fibroblasts upon interleukin (IL)-4, IL-13 and tumour necrosis factor (TNF)-α treatment. In this study, we described that prostaglandin E synthase (PTGES) was upregulated in lung fibroblasts after IL-4, IL-13 and TNF-α treatments. PTGES increased α-SMA levels and promoted lung fibroblast cell migration and invasion abilities. Furthermore, PTGES was upregulated in a lung fibrosis rat model invivo. PTGES increased AKT phosphorylation, leading to activation of the HIF-1α-glycolysis pathway in lung fibroblast cells. HIF-1α inhibitor or 2-DG treatments reduced α-SMA expression in recombinant PTGES (rPTGES)-treated lung fibroblast cells. Targeting PGE2 signalling in PTGES-overexpressing cells by a PTGES inhibitor reduced α-SMA expression. In conclusion, the results of this study demonstrate that PTGES increases the expression of myofibroblast marker via HIF-1α-dependent glycolysis and contributes to myofibroblast differentiation.

NaV1.1 contributes to the cell cycle of human mesenchymal stem cells by regulating AKT and CDK2.

Non-excitable cells express sodium voltage-gated channel alpha subunit 1 gene and protein (known as SCN1A and NaV1.1, respectively); however, the functions of NaV1.1 are unclear. In this study, we investigated the role of SCN1A and NaV1.1 in human mesenchymal stem cells (MSCs). We found that SCN1A was expressed in MSCs, and abundant expression of NaV1.1 was observed in the endoplasmic reticulum; however, this expression was not found to be related to Na+ currents. SCN1A-silencing reduced MSC proliferation and delayed the cell cycle in the S phase. SCN1A silencing also suppressed the protein levels of CDK2 and AKT (herein referring to total AKT), despite similar mRNA expression, and inhibited AKT phosphorylation in MSCs. A cycloheximide-chase assay showed that SCN1A-silencing induced CDK2 but not AKT protein degradation in MSCs. A proteolysis inhibition assay using epoxomicin, bafilomycin A1 and NH4Cl revealed that both the ubiquitin-proteasome system and the autophagy and endo-lysosome system were irrelevant to CDK2 and AKT protein reduction in SCN1A-silenced MSCs. The AKT inhibitor LY294002 did not affect the degradation and nuclear localization of CDK2 in MSCs. Likewise, the AKT activator SC79 did not attenuate the SCN1A-silencing effects on CDK2 in MSCs. These results suggest that NaV1.1 contributes to the cell cycle of MSCs by regulating the post-translational control of AKT and CDK2.

Protective Effects of Sesame Glycoproteins on Ultraviolet-Induced Skin Aging: In Vitro and In Vivo Studies.

Ultraviolet (UV) radiation is a primary factor in skin photoaging, leading to wrinkles, reduced elasticity, and pigmentation changes due to damage to cellular DNA, proteins, and lipids. Glycoproteins from sesame cake (SPE) have potential protective effects against UV-induced skin aging. This study investigated the anti-photoaging effects of SPE on UV-induced damage in human keratinocyte HaCaT cells and SKH-1 hairless mice. SPE was evaluated for its ability to mitigate UV-induced damage in HaCaT cells by assessing MMP-1 protein and mRNA expression levels, as well as the activity of transcription factors AP-1 and NF-κB. The phosphorylation of AKT and MAPK pathways was also analyzed. In vivo, SKH-1 hairless mice were exposed to UV radiation, and the effects of SPE on wrinkle formation and skin structure were assessed by measuring wrinkle length, area, and volume. SPE significantly inhibited UV-induced MMP-1 protein and mRNA expression in HaCaT cells, indicating suppression of AP-1 and NF-κB transcription factors involved in MMP-1 production. Additionally, SPE reduced UV-induced phosphorylation of AKT and MAPK pathways. In SKH-1 hairless mice, SPE treatment led to significant reductions in wrinkle length, area, and volume, preserving skin structure in UV-exposed mice. The findings demonstrate that SPE has protective effects against UV-induced photoaging by inhibiting key molecular pathways associated with skin aging. SPE shows promise as a natural anti-photoaging agent, providing a foundation for future skincare product development. Further studies are warranted to explore the molecular mechanisms in detail and to validate these effects through clinical trials.

Anticancer Effects of BAF312 (Siponimod) in Epithelial Ovarian Cancer.

Epithelial ovarian cancer (EOC) is a lethal disease that is the fifth leading cause of cancer-related death in women. BAF312 (siponimod) is a potent and selective sphingosine-1-phosphate (S1P) receptor modulator that has been approved as a treatment for multiple sclerosis. In addition to its immunomodulatory action, BAF312 shows preclinical antitumor effects in several cancer types. This study sought to determine whether BAF312 had anticancer properties against EOC using in vitro and in vivo models. EOC cell lines A2780, SKOV3ip1, A2780-CP20, and SKOV3-TR were treated with BAF312 and tested for cell proliferation, apoptosis, and migration using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, fluorescence-activated cell sorting, and migration assays. We investigated the expression of sphingosine-1-phosphate receptor 1 (S1PR1) in most EOC cell lines through western blot analysis. To investigate potential mechanisms, western blot analysis was used to assess the expression of AKT serine/threonine kinase 1 (AKT) and extracellular-regulated kinase (ERK) after BAF312 treatment. We also created poly(D,L-lactide-co-glycolide) nanoparticles encapsulating BAF312 (PLGA-NP-BAF312) for in vivo therapy. The average size and zeta potential of PLGA-NP-BAF312 were determined using dynamic light scattering. The therapeutic efficacy of PLGA-NP-BAF312 was tested in an A2780 tumor-bearing orthotopic mouse model of EOC. S1PR1 was overexpressed in most EOC cell lines. BAF312 significantly reduced cell proliferation and migration while inducing significant apoptosis in all EOC cell lines. PLGA-NP-BAF312 treatment significantly reduced tumor weights in A2780 tumor-bearing mice. Furthermore, the anticancer effects of BAF312 were associated with reduced phosphorylation of ERK and AKT. Our findings show that BAF312 has significant anticancer effects in EOC cells by inhibiting the ERK and AKT pathways, and might potentially be used to treat EOCs.

Murine neonatal cardiac regeneration depends on Insulin-like growth factor 1 receptor signaling.

Unlike adult mammals, the hearts of neonatal mice possess the ability to completely regenerate from myocardial infarction (MI). This observation has sparked vast interest in deciphering the potentially lifesaving and morbidity-reducing mechanisms involved in neonatal cardiac regeneration. In mice, the regenerative potential is lost within the first week of life and coincides with a reduction of Insulin-like growth factor 1 receptor (Igf1r) expression in the heart. Igf1r is a well-known regulator of cardiomyocyte maturation and proliferation in neonatal mice. To test the role of Igf1r as a pivotal factor in cardiac regeneration, we knocked down (KD) Igf1r specifically in cardiomyocytes using recombinant adeno-associated virus (rAAV) delivery and troponin T promotor driven shRNAmirs. Cardiomyocyte specific Igf1r KD versus control mice were subjected to experimental MI by permanent ligation of the left anterior descending artery (LAD). Cardiac functional and morphological data were analyzed over a 21-day period. Neonatal Igf1r KD mice showed reduced systolic cardiac function and increased fibrotic cardiac remodeling 21days post injury. This cardiac phenotype was associated with reduced cardiomyocyte nuclei mitosis and decreased AKT and ERK phosphorylation in Igf1r KD, compared to control neonatal mouse hearts. Our in vivo murine data show that Igf1r KD shifts neonatal cardiac regeneration to a more adult-like scarring phenotype, identifying cardiomyocyte-specific Igf1r signaling as a crucial component of neonatal cardiac regeneration.

Sinapine suppresses ROS-induced C2C12 myoblast cell death through MAPK and autophagy pathways.

Oxidative stress in skeletal muscle can lead to muscle atrophy through reactive oxygen species (ROS)-induced damage and cell death. tert-Butyl hydroperoxide (TBHP), an exogenous ROS generator, induces oxidative stress and cell death in various cells. Sinapine from cruciferous plants possesses beneficial effects, but its role in protecting skeletal muscle cells against ROS-induced cell death remains unclear. This study demonstrates that sinapine pretreatment significantly reduced TBHP-induced cell death and ROS accumulation in a dose-dependent manner. TBHP activated mitogen-activated protein kinase (MAPK) pathways including Akt, p38, and JNK, and triggered autophagy. Sinapine suppressed the phosphorylation of Akt, MEK3/6, p38, MEK4, and JNK, and modulated key autophagy markers. Notably, the co-treatment of MAPK inhibitors attenuated TBHP-induced cell death and LC3B-II accumulation. These findings suggest that sinapine is a promising phytochemical for mitigating oxidative stress-mediated muscle injury, offering potential therapeutic strategies for maintaining skeletal muscle homeostasis and addressing muscle-related pathologies.

Progranulin enhances the engraftment of transplanted human iPS cell-derived cerebral neurons.

Cerebral organoids (COs) in cell replacement therapy offer a viable approach to reconstructing neural circuits for individuals suffering from stroke or traumatic brain injuries. Successful transplantation relies on effective engraftment and neurite extension from the grafts. Earlier research has validated the effectiveness of delaying the transplantation procedure by 1 week. Here, we hypothesized that brain tissues 1 week following a traumatic brain injury possess a more favorable environment for cell transplantation when compared to immediately after injury. We performed a transcriptomic comparison to differentiate gene expression between these 2 temporal states. In controlled in vitro conditions, recombinant human progranulin (rhPGRN) bolstered the survival rate of dissociated neurons sourced from human induced pluripotent stem cell-derived COs (hiPSC-COs) under conditions of enhanced oxidative stress. This increase in viability was attributable to a reduction in apoptosis via Akt phosphorylation. In addition, rhPGRN pretreatment before in vivo transplantation experiments augmented the engraftment efficiency of hiPSC-COs considerably and facilitated neurite elongation along the host brain's corticospinal tracts. Subsequent histological assessments at 3 months post-transplantation revealed an elevated presence of graft-derived subcerebral projection neurons-crucial elements for reconstituting neural circuits-in the rhPGRN-treated group. These outcomes highlight the potential of PGRN as a neurotrophic factor suitable for incorporation into hiPSC-CO-based cell therapies.

CircLIFRSA/miR-1305/PTEN axis attenuates malignant cellular processes in non-small cell lung cancer by regulating AKT phosphorylation

BackgroundNon-small cell lung cancer (NSCLC) is typically diagnosed at advanced stages, which limits the effectiveness of therapeutic interventions. The present study aimed to explore the role of the newly identified circLIFRSA in the PTEN/AKT signaling pathway and its involvement in the malignant processes of NSCLC.MethodsCircLIFRSA expression was identified through microarray analysis, and its levels in NSCLC samples were quantified by RT-qPCR. The impact of circLIFRSA on cell growth, proliferation, apoptosis, and cell cycle were evaluated by MTT assay, colony formation assay, and flow cytometry. Additionally, Western blotting was employed to analyze the expression of PTEN and phosphorylated AKT (pAKT) in NSCLC cells.ResultsThe expression of circLIFRSA was found to be significantly reduced in NSCLC cells and tissues. This downregulation correlated with various clinicopathological characteristics and indicated its potential as an early diagnostic biomarker for NSCLC. Importantly, circLIFRSA was shown to inhibit cell growth and proliferation while promoting apoptosis in NSCLC cells. Mechanically, circLIFRSA was found to attenuate the malignant processes of NSCLC cells via the miR-1305/PTEN axis and the suppression of AKT phosphorylation.ConclusionsThese findings indicate that circLIFRSA/miR-1305/PTEN axis attenuates malignant processes by regulating AKT phosphorylation, and provide new insights into the potential of circLIFRSA as a biomarker for early diagnosis and as a promising therapeutic target in NSCLC.

A novel protein SPECC1-415aa encoded by N6-methyladenosine modified circSPECC1 regulates the sensitivity of glioblastoma to TMZ

BackgroundCircular RNAs (circRNAs) can influence a variety of biological functions and act as a significant role in the progression and recurrence of glioblastoma (GBM). However, few coding circRNAs have been discovered in cancer, and their role in GBM is still unknown. The aim of this study was to identify coding circRNAs and explore their potential roles in the progression and recurrence of GBM.MethodsCircSPECC1 was screened via circRNAs microarray of primary and recurrent GBM samples. To ascertain the characteristics and coding ability of circSPECC1, we conducted a number of experiments. Afterward, through in vivo and in vitro experiments, we investigated the biological functions of circSPECC1 and its encoded novel protein (SPECC1-415aa) in GBM, as well as their effects on TMZ sensitivity.ResultsBy analyzing primary and recurrent GBM samples via circRNAs microarray, circSPECC1 was found to be a downregulated circRNA with coding potential in recurrent GBM compared with primary GBM. CircSPECC1 suppressed the proliferation, migration, invasion, and colony formation abilities of GBM cells by encoding a new protein known as SPECC1-415aa. CircSPECC1 restored TMZ sensitivity in TMZ-resistant GBM cells by encoding the new protein SPECC1-415aa. The m6A reader protein IGF2BP1 can bind to circSPECC1 to promote its expression and stability. Mechanistically, SPECC1-415aa can bind to ANXA2 and competitively inhibit the binding of ANXA2 to EGFR, thus resulting in the inhibition of the phosphorylation of EGFR (Tyr845) and its downstream pathway protein AKT (Ser473). In vivo experiments showed that the overexpression of circSPECC1 could combine with TMZ to treat TMZ-resistant GBM, thereby restoring the sensitivity of TMZ-resistant GBM to TMZ.ConclusionsCircSPECC1 was downregulated in recurrent GBM compared with primary GBM. The m6A reader protein IGF2BP1 could promote the expression and stability of circSPECC1. The sequence of SPECC1-415aa, which is encoded by circSPECC1, can inhibit the binding of ANXA2 to EGFR by competitively binding to ANXA2 and inhibiting the phosphorylation of EGFR and AKT, thereby restoring the sensitivity of TMZ-resistant GBM cells to TMZ.

Expression of the apelin system in the porcine pituitary during the oestrous cycle and early pregnancy and the effect of apelin on LH and FSH secretion

Reproductive success requires considerable energy investment. Research has shown that some adipokines, i.e. the hormones produced in adipose tissue, affect reproductive functions by influencing all structures of the hypothalamic-pituitary-ovarian axis. Apelin is a recently identified member of the adipokine family. To the best of the authors' knowledge, this is the first study to investigate the gene and protein expression of the apelin system (the apelin hormone and the apelin receptor, APJ) in the anterior (AP) and posterior (PP) pituitary lobes of the domestic pig during different phases of the oestrous cycle (days 2 to 3, 10 to 12, 14 to 16, and 17 to 19) and in early pregnancy (days 10 to 11, 12 to 13, 15 to 16, and 27 to 28). It was also assumed that apelin participates in the regulation of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion and influences Akt, MAPK/Erk1/2, and AMPK signalling pathways in the AP during the oestrous cycle. Apelin, APJ mRNAs and proteins were detected in both pituitary lobes. Apelin was identified in gonadotropes, somatotropes, lactotropes, and thyrotropes. The study also revealed that apelin and APJ mRNA/protein levels fluctuate during the oestrous cycle and early gestation. Apelin affects basal, GnRH- and/or insulin-stimulated gonadotropin secretion in some phases of the cycle, as well as the phosphorylation of Akt, MAPK/Erk1/2, and AMPK proteins in AP cells. These findings suggest that apelin may be produced locally in the pituitary and that this gland is receptive to apelin's action. The study also suggest that apelin may influence female reproductive functions by controlling the release of LH and FSH from AP cells, and that it affects Akt, MAPK/Erk1/2, and AMPK signalling pathways.

Nifuratel Induces Triple-Negative Breast Cancer Cell G2/M Phase Block and Apoptosis by Regulating GADD45A.

(1) Background: Nifuratel (NF113), derived from nitrofuran, has a specific anti-tumor effect. However, the potential mechanisms of NF113 in triple-negative breast cancer remain unknown. (2) Methods: In the study, CCK8 assay and colony formation assays were used to evaluate the inhibition effect of NF113 on cell proliferation. Apoptosis and cell cycle distribution were tested by flow cytometry. The mechanism of NF113's anti-tumor effect was predicted by transcriptome sequencing and verified by using PCR and Western blot experiments. Breast cancer organoids constructed from the patient-derived tumor xenograft model and the MDA-MB-468 xenograft mouse model were established to evaluate the effect of NF113. (3) Results: Our study showed that NF113 had an anti-tumor effect on triple-negative breast cancer both in vitro and in vivo. NF113 also induced apoptosis and G2/M phase arrest in triple-negative breast cancer cells. Our experimental data further verified that NF113 reduced GADD5A mRNA and protein expression, which were significantly upregulated in breast cancer, with downstream CDC25C and AKT phosphorylation changes. (4) Conclusions: Our data provided compelling evidence that NF113 inhibited breast cancer growth via upregulating GADD45A. Conclusion: NF113 was able to exert inhibitory effects on the proliferation of triple-negative breast cancer in vivo and in vitro, which may induce G2/M phase arrest via the GADD45A/CyclinB/CDK1 pathway and apoptosis via GADD45A/JNK/P38.

Fractalkine/CX3CR1 axis is critical for neuroprotection induced by hypoxic postconditioning against cerebral ischemic injury

Microglial activation-mediated neuroinflammation is a major contributor to neuronal damage after cerebral ischemia. The Fractalkine (FKN)/CX3C chemokine receptor 1 (CX3CR1) axis plays a critical role in regulating microglial activation and neuroinflammation. The aim of this study is to ascertain the role and mechanism of FKN/CX3CR1 axis in hypoxic postconditioning (HPC)-induced anti-inflammatory and neuroprotective effects on transient global cerebral ischemia (tGCI). We found that HPC suppressed microglial activation and alleviated neuroinflammation in hippocampal CA1 after tGCI. Meanwhile, HPC upregulated the expression of FKN and CX3CR1 in neurons, but it downregulated the expression of CX3CR1 in glial cells after tGCI. In addition, the overexpression of FKN induced by the administration of FKN-carried lentivirus reduced microglial activation and inhibited neuroinflammation in CA1 after tGCI. Furthermore, silencing CX3CR1 with CX3CRi-carried lentivirus in CA1 after tGCI suppressed microglial activation and neuroinflammation and exerted neuroprotective effects. Finally, the overexpression of FKN caused a marked increase of neuronal CX3CR1 receptors, upregulated the phosphorylation of Akt, and reduced neuronal loss of rats in CA1 after tGCI. These findings demonstrated that HPC protected against neuronal damage in CA1 of tGCI rats through inhibiting microglial activation and activating Akt signaling pathway via FKN/CX3CR1 axis.

Exploring the Potential Effects and Mechanism of Astragalus Membranaceus in Treating Ischemic Heart Failure Based on Network Pharmacology and Experimental Verification.

Astragalus membranaceus (AM) is a traditional Chinese medicine that has been clinically utilized as an adjunctive therapy for the treatment of myocardial ischemia and heart failure; however, its precise molecular mechanism of action remains unknown. This study aims to investigate the potential pharmacological effects and molecular mechanism of AM in the treatment of ischemic heart failure (IHF) using network pharmacology methods, molecular docking technology, and in vitro experiments. The active components and targets of AM were obtained from the TCMSP databases, while the disease targets of IHF were retrieved from GeneCards and OMIM databases. The analysis of overlapping targets between AM and IHF mainly included active compounds-targets network, PPI network, and GO and KEGG enrichment analysis. The association between active compounds and target proteins was verified through molecular docking. Additionally, an in vitro experimental model was used to evaluate the accuracy of the forecast results. The network pharmacological analysis revealed that quercetin, kaempferol, 7-Omethylisomucronulatol, formononetin, and isorhamnetin were the core active components of AM in treating IHF. The core targets included AKT1, IL6, IL1B, PTGS2, CASP3, MMP9, and HIF1A. The molecular docking results demonstrated a strong binding affinity between these active components and targets. The KEGG pathway analysis suggested that the PI3K-AKT signaling pathway might play a central role in mediating AM's therapeutic effects on IHF. In vitro experiments demonstrated that AM treatment enhanced cell viability, reduced heart failure biomarkers, and suppressed cell apoptosis. Furthermore, the western blot analyses indicated that AM treatment effectively regulated AKT1 phosphorylation in an experimental model of IHF. Through integrated network pharmacological analysis, molecular docking technology, and in vitro experimental validation, it was demonstrated that AM can effectively mitigate IHF through activating PI3K-AKT signaling pathway. These findings significantly advance our understanding of the molecular mechanisms in IHF treatment and contribute further to promoting the clinical application of AM.

Functional activation of the AKT-mTOR signalling axis in a real-world metastatic breast cancer cohort.

Mutations of the PIK3CA/AKT/mTOR axis are common events in metastatic breast cancers (MBCs). This study was designed to evaluate the extent to which genetic alterations of the PIK3CA/AKT/mTOR can predict protein activation of this signalling axis in MBCs. Molecular profiles were generated by CLIA-certified laboratories from a real-world evidence cohort of 171 MBC patients. Genetic alterations of the PIK3CA pathway were measured using next-generation sequencing. Activation levels of AKT and downstream signalling molecules were quantified using two orthogonal proteomic methods. Protein activity was correlated with underlying genomic profiles and response to CDK4/6 inhibition in combination with endocrine treatment (ET). Oncogenic alterations of the PIK3CA/AKT/PTEN pathway were identified in 49.7% of cases. Genomic profiles emerged as poor predictors of protein activity (AUC:0.69), and AKT phosphorylation levels mimicked those of mutant lesions in 76.9% of wild-type tumours. High phosphorylation levels of the PI3K/AKT/mTOR downstream target p70S6 Kinase (T389) were associated with shorter PFS in patients treated with CDK4/6 inhibitors in combination with ET (HR:4.18 95%CI:1.19-14.63); this association was not seen when patients were classified by mutational status. Phosphoprotein-based measurements of drug targets and downstream substrates should be captured along with genomic information to identify MBCs driven by the PI3K/AKT/mTOR signalling.

Gastrodin improves microglia-mediated inflammatory response after hypoxic-ischemic brain damage in neonatal rats via PI3K/AKT pathway

To investigate the mechanism of gastrodin for inhibiting microglia-mediated inflammation after hypoxicischemic brain damage (HIBD) in neonatal rats. Thirty-nine 3-day-old SD rats were randomly divided into sham group, HIBD group and gastrodin treatment group. Western blotting was used to detect the expressions of TNF-α, IL-1β, IL-10 and TGF- β1 in the corpus callosum of the rats. The potential targets of gastrodin for treatment of HIBD were screened by network pharmacology analysis. The expressions of PI3K/AKT signaling pathway proteins following HIBD-induced microglial activation in the rats and in cultured microglial BV-2 cells with oxygen-glucose deprivation (OGD) were detected with Western blotting. The effects of LY294002 (a specific inhibitor of the PI3K/AKT pathway) and gastrodin on TNF-α and TGF-β1 mRNA levels in BV-2 cells with OGD was detected with RT-qPCR. In the neonatal rats with HIBD, gastrodin treatment significantly decreased TNF-α and IL-1β expressions and enhanced IL-10 and TGF-β1 expressions in the ischemic corpus callosum. Network pharmacology analysis showed significant enrichment of the PI3K/AKT signaling pathway and a strong binding between gastrodin and PI3K. Gastrodin significantly promoted PI3K and AKT phosphorylation in neonatal rats with HIBD and in BV-2 cells exposed to OGD. In BV-2 cells with OGD, gastrodin obviously suppressed OGD-induced increase of TNF-α and reduction of TGF-β1 mRNA expressions, and this effect was strongly attenuated by LY294002 treatment. Gastrodin can inhibit microglia-mediated inflammation in neonatal rats with HIBD by regulating the PI3K/AKT signaling pathway.

Phospholipase Cδ-4 (PLCδ4) Acts as a Nuclear Player to Influence Cyclin B Expression in the Embryonal Rhabdomyosarcoma Cell Lines RD and A204.

Rhabdomyosarcoma (RMS), the most common form of sarcoma typical of pediatric age, arises from the malignant transformation of the mesenchymal precursors that fail to differentiate into skeletal muscle cells. Here, we investigated whether the protein phospholipase C δ4 (PLCδ4), a member of the PLC family involved in proliferation and senescence mechanisms of mesenchymal stromal stem cells, may play a role in RMS. Our molecular and morpho-functional data reveal that PLCδ4 is highly expressed in the fusion-negative, p53-positive, SMARCB1 heterozygous mutated embryonal RMS (ERMS) cell line A204, while it is poorly expressed in the ERMS cell lines RD (fusion-negative, MYC amplification, N-RAS (Q61H), homozygous mutated p53) and Hs729 (homozygous mutated p53) and the alveolar rhabdosarcoma (ARMS) cell line SJCRH30 (RH30; fusion positive, heterozygous mutated RARA, polyheterozygous mutated p53). To characterize the role of PLCδ4, the RD cell line was stably transfected with wild-type PLCδ4 (RD/PLCδ4). Overexpressed PLCδ4 mainly localized to the nucleus in RD cells and contributed to the phosphorylation of PRAS40 (T246), Chk2(T68), WNK1(T60), and Akt 1/273 (S473), as revealed by proteome profiler array analysis. Overexpression of PLCδ4 in RD cells enhanced cyclin B1 expression and resulted in G2/M-phase cell cycle arrest. In contrast, siRNA-mediated knockdown of PLCδ4 in A204 cells resulted in reduced cyclin B1 expression. Our study identifies a novel role for nuclear PLCδ4 as a regulator of cyclin B1 via Akt-dependent phosphorylation. The modulation of PLCδ4 expression and its downstream targets could represent a crucial signaling pathway to block embryonal RMS cell proliferation.

Ding-Zhi-Xiao-Wan decoction alleviates mitochondrial autophagy in vascular dementia mice via the PI3K/Akt/mTOR pathway

BackgroundVascular dementia (VD) is a common and diverse group of neurological disorders. The cause of cognitive impairment symptoms in VD is mainly cerebrovascular lesions caused by various factors. Ding-Zhi-Xiao-Wan decoction (DZXW) is a classical Chinese medicine compound for treating clinical manifestations of dementia-like conditions. Our preliminary study determined that the volatile oil component (β-asarone) of Acorus tatarinowii Schott in DZXW has a significant neuroprotective effect. However, there is limited documentation on the effects of DZXW in treating VD. MethodsThe analysis of medicinal chemistry, potential targets, and mechanisms of DZXW for treating VD started with network pharmacology. To create the VD model, bilateral carotid artery ligation was performed. Normal saline or DZXW was administered to Institute of Cancer Research mice via gavage daily for 28 days. The Morris water maze test was used to evaluate the learning and memory abilities of the mice post-treatment. To validate potential targets and efficacy mechanisms identified through network pharmacology, qualitative real-time PCR and western blotting were employed. Further examination of histopathological and ultrastructural changes in the hippocampal region was conducted using hematoxylin-eosin and Nissl staining, immunofluorescence, and transmission electron microscopy. ResultsThe analysis using network pharmacology showed that there was a connection between the key targets associated with the components of DZXW, there were 836 targets of ginseng, 305 targets of Acorus tatarinowii Schott, 394 targets of Poria cocos, and 228 targets of Polygala tenuifolia; and 4671 targets related to VD. In particular, 53 targets are at the intersection of DZXW and VD. The results of this experiment confirmed that DZXW not only attenuated brain damage and its induced learning memory deficits in the VD model but also had some antioxidant effects. Furthermore, DZXW down-regulated VD-induced CASP3, EGFR, PTGS2, ESR1, and HSP90AA1 mRNA expression. It was confirmed that DZXW enhanced the phosphorylation of PI3K, Akt, and mTOR. Additionally, DZXW was demonstrated to elevate the protein levels of p62 and TOM20 while reducing the protein expression of mitochondrial autophagy markers such as PINK1, PARKIN, Beclin-1, and LC3. ConclusionsDZXW impedes mitochondrial autophagy through the activation of the PI3K/Akt/mTOR signaling pathway. This leads to the reduction of mitochondrial injury in nerve cells triggered by VD-induced tissue hypoglycemia and hypoxia, ultimately enhancing cognitive function and memory retention.

MG53's non-physiologic interaction with insulin receptor: lack of effect on insulin-stimulated Akt phosphorylation in muscle, heart and liver tissues.

MG53's known function in facilitating tissue repair and anti-inflammation has broad applications to regenerative medicine. There is controversy regarding MG53's role in the development of type 2 diabetes mellitus. This study aims to address this controversy - whether MG53's myokine function contributes to inhibition of insulin signaling in muscle, heart, and liver tissues. We determined the binding affinity of the recombinant human MG53 (rhMG53) to the insulin receptor extracellular domain (IR-ECD) and found low affinity of interaction with Kd (>480 nM). Using cultured C2C12 myotubes and HepG2 cells, we found no effect of rhMG53 on insulin-stimulated Akt phosphorylation (p-Akt). We performed in vivo assay with C57BL/6J mice subjected to insulin stimulation (1 U/kg, intraperitoneal injection) and observed no effect of rhMG53 on insulin-stimulated p-Akt in muscle, heart and liver tissues. Overall, our data suggest that rhMG53 can bind to the IR-ECD, however has a low likelihood of a physiologic role, as the Kd for binding is ~10,000 higher than the physiologic level of MG53 present in the serum of rodents and humans (~10 pM). Our findings question the notion proposed by Xiao and colleagues - whether targeting circulating MG53 opens a new therapeutic avenue for type 2 diabetes mellitus and its complications.

The improvement of modified Si-Miao granule on hepatic insulin resistance and glycogen synthesis in type 2 diabetes mellitus involves the inhibition of TNF-α/JNK1/IRS-2 pathway: network pharmacology, molecular docking, and experimental validation

BackgroundModified Si-Miao granule (mSMG), a traditional Chinese medicine, is beneficial for T2DM and insulin resistance (IR), but the underlying mechanism remains unknown.MethodsUsing network pharmacology, we screened the compounds of mSMG and identified its targets and pathway on hepatic IR in T2DM. Using molecular docking, we identified the affinity between the compounds and hub target TNF-α. Then these were verified in KK-Ay mice and HepG2 cells.Results50 compounds and 170 targets of mSMG against IR in T2DM were screened, and 9 hub targets such as TNF and MAPK8 were identified. 170 targets were mainly enriched in insulin resistance and TNF pathway, so we speculated that mSMG might act on TNF-α, JNK1 and then regulate insulin signaling to mitigate IR. Experimental validation proved that mSMG ameliorated hyperglycemia, IR, and TNF-α, enhanced glucose consumption and glycogen synthesis, relieved the phosphorylation of JNK1 and IRS-2 (Ser388), and elevated the phosphorylation of Akt (Ser473) and GSK-3β (Ser9) and GLUT2 expression in KK-Ay mice. Molecular docking further showed berberine from mSMG had excellent binding capacity with TNF-α. Then, in vitro validation experiments, we found that 20% mSMG-MS or 50 μM berberine had little effect in IR-HepG2 cell viability, but significantly increased glucose consumption and glycogen synthesis and regulated TNF-α/JNK1/IRS-2 pathway.ConclusionNetwork pharmacology and molecular docking help us predict potential mechanism of mSMG and further guide experimental validation. mSMG and its representative compound berberine improve hepatic IR and glycogen synthesis, and its mechanism may be related to the inhibition of TNF-α/JNK1/IRS-2 pathway.