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Scientific Reports Jun 2022Insulin exerts positive inotropic effects on cardiac muscle; however, the relationship between cardiac contractility and phosphoinositol-3-kinase/Akt (PI3K/Akt)...
Insulin exerts positive inotropic effects on cardiac muscle; however, the relationship between cardiac contractility and phosphoinositol-3-kinase/Akt (PI3K/Akt) activation remains unclear. We hypothesized that the positive inotropic effects of insulin are dose-dependent and mediated via the PI3K/Akt pathway in isolated normal rat hearts. The Institutional Animal Investigation Committee approved the use of hearts excised from rats under pentobarbital anesthesia. The hearts were perfused at a constant pressure using the Langendorff technique. After stabilization (baseline), the hearts were randomly divided into the following four insulin (Ins) groups: 1) Ins0 (0 IU/L), 2) Ins0.5 (0.5 IU/L), 3) Ins5 (5 IU/L), and 4) Ins50 (50 IU/L) (n = 8 in each group). To clarify the role of the PI3K/Akt pathway in insulin-dependent inotropic effects, we also treated the insulin groups with the PI3K inhibitor wortmannin (InsW): 5) InsW0 (0 IU/L), 6) InsW0.5 (0.5 IU/L), 7) InsW5 (5 IU/L), and 8) InsW50 (50 IU/L). Hearts were perfused with Krebs-Henseleit buffer solution with or without wortmannin for 10 min, followed by 20 min perfusion with the solution containing each concentration of insulin. The data were recorded as the maximum left ventricular derivative of pressure development (LV dP/dt max). Myocardial p-Akt levels were measured at 3 min, 5 min, and at the end of the perfusion. In the Ins groups, LV dP/dt max in Ins5 and Ins50 increased by 14% and 48%, respectively, 3 min after insulin perfusion compared with the baseline. Tachyphylaxis was observed after 10 min in the Ins5 and Ins50 treatment groups. Wortmannin partially inhibited the positive inotropic effect of insulin; although insulin enhanced p-Akt levels at all time points compared with the control group, this increase was suppressed in the presence of wortmannin. The positive inotropic effect of insulin is dose-dependent and consistent with Akt activation. This effect mediated by high doses of insulin on cardiac tissue was temporary and caused tachyphylaxis, potentially triggered by Akt overactivation, which leads beta 1 deactivation.
Topics: Animals; Heart; Insulin; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Rats; Wortmannin
PubMed: 35697740
DOI: 10.1038/s41598-022-14092-2 -
Frontiers in Pharmacology 2022Magnolia volatile oil (MVO) is a mixture mainly containing eudesmol and its isomers. This study was to investigate the vasorelaxant effects and the underlying mechanism...
Magnolia volatile oil (MVO) is a mixture mainly containing eudesmol and its isomers. This study was to investigate the vasorelaxant effects and the underlying mechanism of MVO in rat thoracic aortas. The present study combined gas chromatography-mass spectrometry (GC-MS) and network pharmacology analysis with experiments to clarify the mechanisms of MVO against vessel contraction. A compound-target network, compound-target-disease network, protein-protein interaction network, compound-target-pathway network, gene ontology, and pathway enrichment for hypertension were applied to identify the potential active compounds, drug targets, and pathways. Additionally, the thoracic aortic rings with or without endothelium were prepared to explore the underlying mechanisms. The roles of the PI3K-Akt-NO pathways, neuroreceptors, K channels, and Ca channels on the vasorelaxant effects of MVO were evaluated through the rat thoracic aortic rings. A total of 29 compounds were found in MVO, which were identified by GC-MS, of which 21 compounds with a content of more than 0.1% were selected for further analysis. The network pharmacology research predicted that beta-caryophyllene, palmitic acid, and (+)-β-selinene might act as the effective ingredients of MVO for the treatment of hypertension. Several hot targets, mainly involving TNF, CHRM1, ACE, IL10, PTGS2, REN, and F2, and pivotal pathways, such as the neuroactive ligand-receptor interaction, the calcium signaling pathway, and the PI3K-Akt signaling, were responsible for the vasorelaxant effect of MVO. As expected, MVO exerted a vasorelaxant effect on the aortic rings pre-contracted by KCl and phenylephrine in an endothelium-dependent and non-endothelium-dependent manner. Importantly, a pre-incubation with indomethacin (Indo), -nitro--arginine methyl ester, methylene blue, wortmannin, and atropine sulfate as well as 4-aminopyridione diminished MVO-induced vasorelaxation, suggesting that the activation of the PI3K-Akt-NO pathway and K channel were involved in the vasorelaxant effect of MVO, which was consistent with the results of the Kyoto Encyclopedia of Genes and the Genomes. Additionally, MVO could significantly inhibit Ca influx resulting in the contraction of aortic rings, revealing that the inhibition of the calcium signaling pathway exactly participated in the vasorelaxant activity of MVO as predicted by network pharmacology. MVO might be a potent treatment of diseases with vascular dysfunction like hypertension. The underlying mechanisms were related to the PI3K-Akt-NO pathway, K pathway, as well as Ca channel, which were predicted by the network pharmacology and verified by the experiments This study based on network pharmacology provided experimental support for the clinical application of MVO in the treatment of hypertension and afforded a novel research method to explore the activity and mechanism of traditional Chinese medicine.
PubMed: 35308213
DOI: 10.3389/fphar.2022.812716 -
Breast Cancer Research : BCR May 2022Opioid and beta-adrenergic receptors are recently shown to cross talk via formation of receptor heterodimers to control the growth and proliferation of breast cancer...
Beta 2 adrenergic receptor and mu opioid receptor interact to potentiate the aggressiveness of human breast cancer cell by activating the glycogen synthase kinase 3 signaling.
BACKGROUND
Opioid and beta-adrenergic receptors are recently shown to cross talk via formation of receptor heterodimers to control the growth and proliferation of breast cancer cells. However, the underlying cell signaling mechanism remained unclear.
METHODS
To determine the effect of the interaction of the two systems in breast cancer, we employed triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468, CRISPR or chemical inhibition or activation of beta-adrenergic receptors (B2AR) and mu-opioid receptors (MOR) gene, and PCR array technology and studied aggressive tumor phenotype and signaling cascades.
RESULTS
We show here that in triple-negative breast cancer cells, the reduction in expression B2AR and MOR by genetic and pharmacological tools leads to a less aggressive phenotype of triple-negative breast cancer cells in vitro and in animal xenografts. Genomic analysis indicates the glycogen synthase kinase 3 (GSK3) pathway as a possible candidate messenger system involved in B2AR and MOR cross talk. GSK3 inactivation in MDA-MB-231 and MDA-MB-468 cells induced similar phenotypic changes as the inhibition of B2AR and/or MOR, while a GSK3 activation by wortmannin reversed the effects of B2AR and/or MOR knockdown on these cells. GSK3 inactivation also prevents B2AR agonist norepinephrine or MOR agonist DAMGO from affecting MDA-MB-231 and MDA-MB-468 cell proliferation.
CONCLUSIONS
These data confirm a role of B2AR and MOR interaction in the control of breast cancer cell growth and identify a possible role of the GSK3 signaling system in mediation of these two receptors' cross talk. Screening for ligands targeting B2AR and MOR interaction and/or the GSK3 system may help to identify novel drugs for the prevention of triple-negative breast cancer cell growth and metastasis.
Topics: Animals; Cell Line, Tumor; Glycogen Synthase Kinase 3; Humans; Receptors, Adrenergic, beta-2; Receptors, Opioid, mu; Signal Transduction; Triple Negative Breast Neoplasms
PubMed: 35568869
DOI: 10.1186/s13058-022-01526-y -
Scientific Reports Mar 2020The two vasoactive hormones, angiotensin II (ANG II; vasoconstrictive) and atrial natriuretic peptide (ANP; vasodilatory) antagonize the biological actions of each...
The two vasoactive hormones, angiotensin II (ANG II; vasoconstrictive) and atrial natriuretic peptide (ANP; vasodilatory) antagonize the biological actions of each other. ANP acting through natriuretic peptide receptor-A (NPRA) lowers blood pressure and blood volume. We tested hypothesis that ANG II plays critical roles in the transcriptional repression of Npr1 (encoding NPRA) and receptor function. ANG II significantly decreased NPRA mRNA and protein levels and cGMP accumulation in cultured mesangial cells and attenuated ANP-mediated relaxation of aortic rings ex vivo. The transcription factors, cAMP-response element-binding protein (CREB) and heat-shock factor-4a (HSF-4a) facilitated the ANG II-mediated repressive effects on Npr1 transcription. Tyrosine kinase (TK) inhibitor, genistein and phosphatidylinositol 3-kinase (PI-3K) inhibitor, wortmannin reversed the ANG II-dependent repression of Npr1 transcription and receptor function. ANG II enhanced the activities of Class I histone deacetylases (HDACs 1/2), thereby decreased histone acetylation of H3K9/14ac and H4K8ac. The repressive effect of ANG II on Npr1 transcription and receptor signaling seems to be transduced by TK and PI-3K pathways and modulated by CREB, HSF-4a, HDACs, and modified histones. The current findings suggest that ANG II-mediated repressive mechanisms of Npr1 transcription and receptor function may provide new molecular targets for treatment and prevention of hypertension and cardiovascular diseases.
Topics: Acetylation; Angiotensin II; Animals; Cyclic AMP Response Element-Binding Protein; Dose-Response Relationship, Drug; Gene Expression Regulation; Heat Shock Transcription Factors; Histone Deacetylases; Histones; Mice; Models, Biological; Protein Binding; Receptors, Atrial Natriuretic Factor; Transcriptional Activation
PubMed: 32152395
DOI: 10.1038/s41598-020-61041-y -
American Journal of Physiology. Cell... Jul 2021The resolution of inflammation is closely linked with tissue repair. Recent studies have revealed that macrophages suppress inflammatory reactions by producing lipid...
The resolution of inflammation is closely linked with tissue repair. Recent studies have revealed that macrophages suppress inflammatory reactions by producing lipid mediators, called specialized proresolving mediators (SPMs); however, the biological significance of SPMs in tissue repair remains to be fully elucidated in the heart. In this study, we focused on maresin-1 (MaR1) and examined the reparative effects of MaR1 in cardiomyocytes. The treatment with MaR1 increased cell size in cultured neonatal rat cardiomyocytes. Since the expression of fetal cardiac genes was unchanged by MaR1, physiological hypertrophy was induced by MaR1. SR3335, an inhibitor of retinoic acid-related orphan receptor α (RORα), mitigated MaR1-induced cardiomyocyte hypertrophy, consistent with the recent report that RORα is one of MaR1 receptors. Importantly, in response to MaR1, cardiomyocytes produced IGF-1 via RORα. Moreover, MaR1 activated phosphoinositide 3-kinase (PI3K)/Akt signaling pathway and wortmannin, a PI3K inhibitor, or triciribine, an Akt inhibitor, abrogated MaR1-induced cardiomyocyte hypertrophy. Finally, the blockade of IGF-1 receptor by NVP-AEW541 inhibited MaR-1-induced cardiomyocyte hypertrophy as well as the activation of PI3K/Akt pathway. These data indicate that MaR1 induces cardiomyocyte hypertrophy through RORα/IGF-1/PI3K/Akt pathway. Considering that MaR1 is a potent resolving factor, MaR1 could be a key mediator that orchestrates the resolution of inflammation with myocardial repair.
Topics: Animals; Cardiomegaly; Cardiotonic Agents; Disease Models, Animal; Docosahexaenoic Acids; Gene Expression Regulation; Insulin-Like Growth Factor I; Male; Mice; Mice, Inbred C57BL; Myocardial Infarction; Myocytes, Cardiac; Nuclear Receptor Subfamily 1, Group F, Member 1; Paracrine Communication; Phosphatidylinositol 3-Kinases; Primary Cell Culture; Proto-Oncogene Proteins c-akt; Pyrimidines; Pyrroles; Rats; Ribonucleosides; Signal Transduction; Sulfonamides; Thiophenes; Wortmannin
PubMed: 34038245
DOI: 10.1152/ajpcell.00568.2020 -
Frontiers in Immunology 2021Dendritic cells (DCs) have the ability to stimulate naïve T cells that coordinate subsequent adaptive response toward an inflammatory response or tolerance depending on...
Dendritic cells (DCs) have the ability to stimulate naïve T cells that coordinate subsequent adaptive response toward an inflammatory response or tolerance depending on the DC differentiation level. Inotodiol, a lanostane triterpenoid found in (wild Chaga mushroom), is a natural compound with a wide range of biological activities. In this study, we investigated whether inotodiol promotes the maturation of bone marrow-derived DCs (BMDCs) and inotodiol-treated BMDCs induce T cell activation. Inotodiol increased the expression of surface maturation markers, including MHC-I, MHC-II, CD86, and CD40, on BMDCs without affecting the production of various cytokines, including TNF-α and IL-12p40 in these cells. T cells primed with inotodiol-treated BMDCs proliferated and produced IL-2, without producing other cytokines, including IL-12p40 and IFN-γ. Injection of inotodiol into mice induced maturation of splenic DCs and IL-2 production, and the administration of inotodiol and inotodiol-treated BMDCs induced the proliferation of adoptively transferred CD8 T cells . The phosphatidylinositol-3-kinase inhibitor wortmannin abrogated the upregulation of Akt phosphorylation and CD86 and MHC-II expression induced by inotodiol. However, inotodiol failed to induce phosphorylation of the IκB kinase and degradation of IκB-α, and increased expression of CD86 induced by inotodiol was not blocked by an IκB kinase inhibitor. These results suggest that inotodiol induces a characteristic type of maturation in DCs through phosphatidylinositol-3-kinase activation independent of NF-κB, and inotodiol-treated DCs enhance T cell proliferation and IL-2 secretion.
Topics: Agaricales; Animals; Bone Marrow Cells; CD8-Positive T-Lymphocytes; Cell Differentiation; Cell Proliferation; Cells, Cultured; Cytokines; Dendritic Cells; Inonotus; Lanosterol; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Molecular Structure; Phosphorylation; Proto-Oncogene Proteins c-akt; RAW 264.7 Cells
PubMed: 33777049
DOI: 10.3389/fimmu.2021.650841 -
Biomedicines Mar 2023Osteopontin (OPN) is a bioactive integrin-binding protein found in high concentrations in milk, where it is present both as a full-length protein and as several...
Osteopontin (OPN) is a bioactive integrin-binding protein found in high concentrations in milk, where it is present both as a full-length protein and as several N-terminally derived fragments. OPN resists gastric digestion, and via interaction with receptors in the gut or by crossing the intestinal barrier into circulation, ingested milk OPN may influence physiological processes. The aim of this study was to investigate OPN interaction with intestinal cells and its transport across models of the intestinal barrier. Immunodetection of OPN incubated with Caco-2 cells at 4 °C and 37 °C showed that OPN binds to the intestinal cells, but it is not internalised. Transepithelial transport was studied using mono- and co-cultures of Caco-2 cells and mucus-producing HT29-MTX cells in transwell membranes. OPN was shown to cross the barrier models in a time-, temperature-, and energy-dependent process inhibited by wortmannin, indicating that the transport takes place via the transcytosis pathway. Analyses of the naturally occurring milk mixture of full-length and N-terminal fragments showed that the N-terminal fragments of OPN bound intestinal cells most effectively and that the fragments were transported across the intestinal membrane models. This suggests that proteolytic processing of OPN increases its biological activity after ingestion.
PubMed: 36979872
DOI: 10.3390/biomedicines11030893 -
BMC Veterinary Research Mar 2023Canine distemper virus (CDV) is one of the most contagious and lethal viruses known to the Canidae, with a very broad and expanding host range. Autophagy serves as a...
BACKGROUND
Canine distemper virus (CDV) is one of the most contagious and lethal viruses known to the Canidae, with a very broad and expanding host range. Autophagy serves as a fundamental stabilizing response against pathogens, but some viruses have been able to evade or exploit it for their replication. However, the effect of autophagy mechanisms on CDV infection is still unclear.
RESULTS
In the present study, autophagy was induced in CDV-infected Vero cells as demonstrated by elevated LC3-II levels and aggregation of green fluorescent protein (GFP)-LC3 spots. Furthermore, CDV promoted the complete autophagic process, which could be determined by the degradation of p62, co-localization of LC3 with lysosomes, GFP degradation, and accumulation of LC3-II and p62 due to the lysosomal protease inhibitor E64d. In addition, the use of Rapamycin to promote autophagy promoted CDV replication, and the inhibition of autophagy by Wortmannin, Chloroquine and siRNA-ATG5 inhibited CDV replication, revealing that CDV-induced autophagy facilitated virus replication. We also found that UV-inactivated CDV still induced autophagy, and that nucleocapsid (N) protein was able to induce complete autophagy in an mTOR-dependent manner.
CONCLUSIONS
This study for the first time revealed that CDV N protein induced complete autophagy to facilitate viral replication.
Topics: Animals; Dogs; Autophagy; Chlorocebus aethiops; Distemper; Distemper Virus, Canine; Dog Diseases; Vero Cells; Virus Replication; Nucleocapsid Proteins
PubMed: 36922800
DOI: 10.1186/s12917-023-03575-7 -
Aging Jan 2022Autophagy is an important regulator of cellular homeostasis and its dysregulation often results in cancer. Aberrant glycosylation induced by oncogenic transformation...
Autophagy is an important regulator of cellular homeostasis and its dysregulation often results in cancer. Aberrant glycosylation induced by oncogenic transformation contributes to tumor invasion and metastasis. In a previous study, we have demonstrated that EpCAM, a glycosylation protein, is associated with cell growth and metastasis in breast cancer. But the effect of EpCAM glycosylation on autophagy is not clear. the precise mechanism of regulation remains largely unknown. In this study, breast cancer cells were transfected with N-glycosylation mutation EpCAM plasmid to express deglycosylated EpCAM. The result showed that deglycosylated EpCAM promoted autophagy in breast cancer cells. We further confirmed this conclusion with the activator (Rapamycin, RAP) and inhibitor (Wortmannin) of autophagy. We also found that deglycosylated EpCAM promoted apoptosis and inhibited proliferation through activating autophagy by suppressing Akt/mTOR signaling pathway in breast cancer cells. These findings represent a novel mechanism by which deglycosylated EpCAM inhibits proliferation by enhancing autophagy of breast cancer cells via PI3K/Akt/mTOR pathway. In conclusion, the combination of autophagy modulation and EpCAM targeted therapy is a promising therapeutic strategy in the treatment of breast cancer.
Topics: Antifungal Agents; Autophagy; Biomarkers; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Epithelial Cell Adhesion Molecule; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Heterocyclic Compounds, 3-Ring; Humans; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Sirolimus; TOR Serine-Threonine Kinases; Wortmannin
PubMed: 34983878
DOI: 10.18632/aging.203795 -
Ecotoxicology and Environmental Safety Jun 2023Due to the rapid production growth and a wide range of applications, safety concerns are being raised about the genotoxic properties of silver nanoparticles (AgNPs). In...
Due to the rapid production growth and a wide range of applications, safety concerns are being raised about the genotoxic properties of silver nanoparticles (AgNPs). In this research, we found AgNPs induced a size-dependent genotoxicity via lysosomal-autophagy dysfunction in human-hamster hybrid (A) cells. Compared with 25 nm and 75 nm particles, 5 nm AgNPs could accentuate the genotoxic responses, including DNA double-strand breaks (DSBs) and multi-locus deletion mutation, which could be significantly enhanced by autophagy inhibitors 3-methyl adenine (3-MA), Bafilomycin A1 (BFA), and cathepsin inhibitors, respectively. The autophagy dysfunction was closely related to the accumulation of 5 nm AgNPs in the lysosomes and the interruption of lysosome-autophagosome fusion. With lysosomal protective agent 3-O-Methylsphingomyelin (3-O-M) and endocytosis inhibitor wortmannin, the reactivation of lysosomal function and the recovery of autophagy significantly attenuated AgNP-induced genotoxicity. Our data provide clear evidence to illustrate the role of subcellular targets in the genotoxicity of AgNPs in mammalian cells, which laid the basis for better understanding the health risk of AgNPs and their related products.
Topics: Animals; Humans; Silver; Metal Nanoparticles; Autophagy; Lysosomes; Sequence Deletion; Mammals
PubMed: 37105094
DOI: 10.1016/j.ecoenv.2023.114947