OBJECTIVE: To investigate the molecular mechanism underlying the anti-hepatic fibrosis activity of ethyl acetate fraction Dicliptera chinensis (L.) Juss. (EDC) in human hepatic stellate cells (HSCs) in vitro and in a carbon tetrachloride (CCl₄)-induced hepatic fibrosis mouse model in vivo. METHODS: For in vitro study, HSCs were pre-treated with platelet-derived growth factor (10 ng/mL) for 2 h to ensure activation and treated with EDC for 24 h and 48 h, respectively. The effect of EDC on HSCs was assessed using cell counting kit-8 assay, EdU staining, transmission electron microscopy, immunofluorescence staining, and Western blot, respectively. For in vivo experiments, mice were intraperitoneally injected with CCl₄ (2 ° L/g, adjusted to a 25% concentration in olive oil), 3 times per week for 6 weeks, to develop a hepatic fibrosis model. Forty 8-week-old male C57BL/6 mice were divided into 4 groups using a random number table (n=10), including control, model, positive control and EDC treatment groups. Mice in the EDC and colchicine groups were intragastrically administered EDC (0.5 g/kg) or colchicine (0.2 mg/kg) once per day for 6 weeks. Mice in the control and model groups received an equal volume of saline. Biochemical assays and histological examinations were used to assess liver damage. Protein expression levels of α -smooth muscle actin (α -SMA) and microtubule-associated protein light chain 3B (LC3B) were measured by Western blot. RESULTS: EDC reduced pathological damage associated with liver fibrosis, downregulated the expression of α -SMA and upregulated the expression of LC3B (P<0.05), both in HSCs and the CCl₄-induced liver fibrosis mouse model. The intervention of bafilomycin A1 and rapamycin in HSCs strongly supported the notion that inhibition of autophagy enhanced α -SMA protein expression levels (P<0.01). The results also found that the levels of phosphoinositide (PI3K), p-PI3K, AKT, p-AKT, mammalian target of rapamycin (mTOR), p-mTOR, and p-p70S6K all decreased after EDC treatment (P<0.05). CONCLUSIONS EDC has anti-hepatic fibrosis activity by inducing autophagy and might be a potential drug to be further developed for human liver fibrosis therapy.
Acetates ; Animals ; Autophagy ; Carbon Tetrachloride ; Hepatic Stellate Cells ; Liver/pathology* ; Liver Cirrhosis/pathology* ; Male ; Mice ; Mice, Inbred C57BL ; Phosphatidylinositol 3-Kinases ; Proto-Oncogene Proteins c-akt/metabolism* ; Ribosomal Protein S6 Kinases, 70-kDa ; Signal Transduction ; TOR Serine-Threonine Kinases/metabolism*

OBJECTIVE: To investigate the effects and mechanisms of Kindlin-2 on uterus development and reproductive capacity in female mice. METHODS: Cdh16-Cre tool mice and Kindlin-2^flox/flox mice were used to construct the mouse model of uterus specific knockout of Kindlin-2, and the effects of Kindlin-2 deletion on uterine development and reproduction capacity of female mice were observed. High expression and knockdown of Kindlin-2 in endometrial cancer cell lines HEC-1 and Ish were used to detect the regulation of mammalian target of rapamycin (mTOR) signaling pathway. In addition, uterine proteins of the female mice with specific knockout of Kindlin-2 and female mice in the control group were extracted to detect the protein levels of key molecules of mTOR signaling pathway and Hippo signaling pathway. RESULTS: The mouse model of uterine specific knockout of Kindlin-2 was successfully constructed. The knockout efficiency of Kindlin-2 in mouse uterus was identified and verified by mouse tail polymerase chain reaction (PCR), Western blot protein identification, immunohistochemical staining (IHC) and other methods. Compared with the control group, the female mice with uterus specific deletion of Kindlin-2 lost weight, seriously impaired reproductive ability, and the number of newborn mice decreased, but the proportion of the female mice and male mice in the newborn mice did not change. Hematoxylin eosin staining (HE) experiment showed that the endometrium of Kindlin-2 knockout group was incomplete and the thickness of uterine wall became thinner. In terms of mechanism, the deletion of Kindlin-2 in endo-metrial cancer cell lines HEC-1 and Ish could downregulate the protein levels of mTOR, phosphorylated mTOR, adenosine monophosphate-activated protein kinase (AMPK), phosphorylated AMPK and phosphorylated ribosomal protein S6 (S6), and the mTOR signal pathway was inhibited. It was found that the specific deletion of Kindlin-2 could upregulate the protein levels of Mps one binding 1 (MOB1) and phosphorylated Yes-associated protein (YAP) in the uterus of the female mice, and the Hippo signal pathway was activated. CONCLUSION Kindlin-2 inhibits the development of uterus by inhibiting mTOR signal pathway and activating Hippo signal pathway, thereby inhibiting the fertility of female mice.
AMP-Activated Protein Kinases/metabolism* ; Adenosine Monophosphate/metabolism* ; Animals ; Cadherins/metabolism* ; Cytoskeletal Proteins/metabolism* ; Endometrium/metabolism* ; Eosine Yellowish-(YS)/metabolism* ; Female ; Hematoxylin/metabolism* ; Hippo Signaling Pathway ; Male ; Mammals/metabolism* ; Mice ; Muscle Proteins ; Ribosomal Protein S6/metabolism* ; Sirolimus/metabolism* ; TOR Serine-Threonine Kinases/metabolism* ; YAP-Signaling Proteins

OBJECTIVE: To investigate the effect of moxibustion on autophagy and amyloid β-peptide_1-42 (Aβ_1-42) protein expression in amyloid precursor protein/presenilin 1 (APP/PS1) double-transgenic mice with Alzheimer's disease (AD). METHODS: After 2-month adaptive feeding, fifty-six 6-month-old APP/PS1 double transgenic AD mice were randomly divided into a model group, a moxibustion group, a rapamycin group and an inhibitor group, 14 mice in each group. Another 14 C57BL/6J mice with the same age were used as a normal group. The mice in the moxibustion group were treated with monkshood cake-separated moxibustion at "Baihui"(GV 20), "Fengfu" (GV 16) and "Dazhui" (GV 14) for 20 min; the mice in the rapamycin group were intraperitoneally injected with rapamycin (2 mg/kg); the mice in the inhibitor group were treated with moxibustion and injection of 1.5 mg/kg 3-methyladenine (3-MA). All the treatments were given once a day for consecutive 2 weeks. The morphology of hippocampal tissue was observed by HE staining; the ultrastructure of hippocampal tissue was observed by transmission electron microscopy; the expression of Aβ_1-42 protein in frontal cortex and hippocampal tissue was detected by immunohistochemistry; the expressions of mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), p70 ribosomal protein S6 kinase (p70S6K) and phosphorylated p70S6K (p-p70S6K) protein in hippocampus were detected by Western blot method. RESULTS: Compared with the normal group, the number of neuron cells was decreased, cells were necrotic and deformed, and autophagy vesicle and lysosome were decreased in the model group. Compared with the model group, the number of neuron cells was increased, cell necrosis was decreased, and autophagy vesicle and lysosome were increased in the moxibustion group and the rapamycin group. Compared with the normal group, the protein expressions of Aβ_1-42, mTOR, p-mTOR, p70S6K and p-p70S6K in the model group were increased (P<0.05); compared with the model group, the protein expressions of Aβ_1-42, mTOR, p-mTOR, p70S6K and p-p70S6K in the moxibustion group, rapamycin group and inhibitor group were decreased (P<0.05); compared with the inhibitor group, the protein expressions of Aβ_1-42, mTOR, p-mTOR, p70S6K and p-p70S6K in the moxibustion group and rapamycin group were decreased (P<0.05); compared with the rapamycin group, the protein expressions of mTOR, p-mTOR, p70S6K and p-p70S6K in the moxibustion group were decreased (P<0.05). CONCLUSION Moxibustion could enhance autophagy in hippocampal tissue of APP/PS1 double transgenic AD mice and reduce abnormal Aβ aggregation in brain tissue, the mechanism may be related to the inhibition of mTOR/p70S6K signaling pathway.
Alzheimer Disease/therapy* ; Amyloid beta-Peptides/genetics* ; Animals ; Autophagy ; Disease Models, Animal ; Hippocampus/metabolism* ; Mammals/metabolism* ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Moxibustion ; Ribosomal Protein S6 Kinases, 70-kDa/pharmacology* ; Signal Transduction ; Sirolimus/pharmacology* ; TOR Serine-Threonine Kinases/metabolism*

Eukaryotic translation initiation factor 4B (eIF4B) plays an important role in mRNA translation initiation, cell survival and proliferation in vitro, but the in vivo function is poorly understood. In this study, via various experimental techniques such as hematoxylin-eosin (HE) staining, flow cytometry, Western blotting, and immunohistochemistry, we investigated the role of eIF4B in mouse embryo development using an eIF4B knockout (KO) mouse model and explored the mechanism. We found that the livers, but not lungs, brain, stomach, or pancreas, derived from eIF4B KO mouse embryos displayed severe pathological changes characterized by enhanced apoptosis and necrosis. Accordingly, high expression of cleaved-caspase 3, and excessive activation of mTOR signaling as evidenced by increased expression and phosphorylation of p70S6K and enhanced phosphorylation of 4EBP1, were observed in mouse embryonic fibroblasts and fetal livers from eIF4B KO mice. These results uncover a critical role of eIF4B in mouse embryo development and provide important insights into the biological functions of eIF4B in vivo.
Animals ; Apoptosis/genetics* ; Caspase 3 ; Eosine Yellowish-(YS) ; Eukaryotic Initiation Factors/metabolism* ; Fibroblasts ; Hematoxylin ; Liver/metabolism* ; Mice ; Ribosomal Protein S6 Kinases, 70-kDa/genetics* ; TOR Serine-Threonine Kinases

OBJECTIVE: To identify potential mutations of the CLS gene in a Chinese pedigree affected with Coffin-Lowry syndrome. METHODS: Whole exome sequencing was applied to detect potential mutation in the proband, and the result was verified by Sanger sequencing. RESULTS: The proband was found to carry a c.966_967delAA (p.Arg323Thr fs*11) deletional mutation in the RPS6KA3 gene. The same mutation was also found in his mother. CONCLUSION The c.966_967delAA (p.Arg323Thr fs*11) deletional mutation of the RPS6KA3 gene probably underlies the disorder in this pedigree.
Asian Continental Ancestry Group ; China ; Coffin-Lowry Syndrome ; genetics ; Humans ; Mutation ; Pedigree ; Ribosomal Protein S6 Kinases, 90-kDa ; genetics ; Sequence Deletion

OBJECTIVE: Astrocyte dysfunctions are related to several central nervous system (CNS) pathologies. Transcriptomic profiling of human mRNAs to investigate astrocyte functions may provide the basic molecular-biological data pertaining to the cellular activities of astrocytes. METHODS: Human Primary astrocytes (HPAs) and human neural stem cell line (HB1.F3) were used for differential digital gene analysis. In this study, a massively parallel sequencing platform, next-generation sequencing (NGS), was used to obtain the digital gene expression (DGE) data from HPAs. A comparative analysis of the DGE from HPA and HB1.F3 cells was performed. Sequencing was performed using NGS platform, and subsequently, bioinformatic analyses were implemented to reveal the identity of the pathways, relatively up- or down-regulated in HPA cells. RESULTS: The top, novel canonical pathways up-regulated in HPA cells than in the HB1.F3 cells were “Cyclins and cell cycle regulation,” “Integrin signaling,” “Regulation of eIF4 and p70S6K signaling,” “Wnt/β-catenin signaling,” “mTOR signaling,” “Aryl hydrocarbon receptor signaling,” “Hippo signaling,” “RhoA signaling,” “Signaling by Rho family GTPases,” and “Glioma signaling” pathways. The down-regulated pathways were “Cell cycle: G1/S checkpoint regulation,” “eIF2 signaling,” “Cell cycle: G2/M DNA damage checkpoint regulation,” “Telomerase signaling,” “RhoGDI signaling,” “NRF2-mediated oxidative stress response,” “ERK/MAPK signaling,” “ATM signaling,” “Pancreatic adenocarcinoma signaling,” “VEGF signaling,” and “Role of CHK proteins in cell cycle checkpoint control” pathways. CONCLUSION: This study would be a good reference to understand astrocyte functions at the molecular level, and to develop a diagnostic test, based on the DGE pattern of astrocytes, as a powerful, new clinical tool in many CNS diseases.
Adenocarcinoma ; Astrocytes ; Cell Cycle ; Cell Cycle Checkpoints ; Central Nervous System ; Central Nervous System Diseases ; Computational Biology ; Diagnostic Tests, Routine ; DNA Damage ; Gene Expression ; High-Throughput Nucleotide Sequencing ; Humans ; Neural Stem Cells ; Oxidative Stress ; Pathology ; Ribosomal Protein S6 Kinases, 70-kDa ; RNA, Messenger

p90 ribosomal S6 kinase (p90RSK), one of the downstream effectors in ERK1/2 pathways, shows high expression in human breast cancer tissues. However, its role in breast cancer development and drug resistance is not fully understood. Here, we demonstrate that Cis-DDP treatment failed to increase cytotoxicity in MDA-MB-231 cells compared to MCF-7 cells and p90RSK activation was involved in Cis-DDP-resistance to MDA-MB-231 cells. In the study, we found that inhibition of p90RSK expression or activation using a small interfering RNA (siRNA) or dominant-negative kinase mutant (DN-p90RSK) plasmid overexpression increased Cis-DDP-induced cytotoxicity of MDA-MB-231 cells, respectively. Mechanistically, we found that Cis-DDP resistance was associated with up-regulation of epithelial growth factor (EGF) expression and EGF treatment induced cancer survival signaling pathway including activation of ERK1/2, p90RSK, and Akt. We also examined the expression of epithelial-mesenchymal transition (EMT)-associated proteins using a reverse transition-quantitative PCR analysis. Cis-DDP treatment induced EMT by increasing the expression levels of N-cadherin, Snail, and Twist, while decreasing the expression levels of E-cadherin. Furthermore, we examined the epithelial marker, Zonula occludens-1 (ZO-1) using immunofluorescence analysis and found that Cis-DDP-inhibited ZO-1 expression was recovered by p90RSK deactivated condition. Therefore, we conclude that Cis-DDP resistance is involved in EMT via regulating the EGF-mediated p90RSK signaling pathway in MDA-MB-231 cells.
Breast Neoplasms ; Cadherins ; Cisplatin ; Drug Resistance ; Epidermal Growth Factor ; Epithelial-Mesenchymal Transition ; Fluorescent Antibody Technique ; Humans ; MCF-7 Cells ; Phosphotransferases ; Plasmids ; Polymerase Chain Reaction ; Ribosomal Protein S6 Kinases, 90-kDa ; RNA, Small Interfering ; Snails ; Triple Negative Breast Neoplasms ; Up-Regulation

During spermatogenesis, developing germ cells that lack the cellular ultrastructures of filopodia and lamellipodia generally found in migrating cells, such as macrophages and fibroblasts, rely on Sertoli cells to support their transport across the seminiferous epithelium. These include the transport of preleptotene spermatocytes across the blood-testis barrier (BTB), but also the transport of germ cells, in particular developing haploid spermatids, across the seminiferous epithelium, that is to and away from the tubule lumen, depending on the stages of the epithelial cycle. On the other hand, cell junctions at the Sertoli cell-cell and Sertoli-germ cell interface also undergo rapid remodeling, involving disassembly and reassembly of cell junctions, which, in turn, are supported by actin- and microtubule-based cytoskeletal remodeling. Interestingly, the underlying mechanism(s) and the involving biomolecule(s) that regulate or support cytoskeletal remodeling remain largely unknown. Herein, we used an in vitro model of primary Sertoli cell cultures that mimicked the Sertoli BTB in vivo overexpressed with the ribosomal protein S6 (rpS6, the downstream signaling protein of mammalian target of rapamycin complex 1 [mTORC1]) cloned into the mammalian expression vector pCI-neo, namely, quadruple phosphomimetic and constitutively active mutant of rpS6 (pCI-neo/p-rpS6-MT) versus pCI-neo/rpS6-WT (wild-type) and empty vector (pCI-neo/Ctrl) for studies. These findings provide compelling evidence that the mTORC1/rpS6 signal pathway exerted its effects to promote Sertoli cell BTB remodeling. This was mediated through changes in the organization of actin- and microtubule-based cytoskeletons, involving changes in the distribution and/or spatial expression of actin- and microtubule-regulatory proteins.
Actins/metabolism* ; Animals ; Blood-Testis Barrier/metabolism* ; Cells, Cultured ; Male ; Mechanistic Target of Rapamycin Complex 1/metabolism* ; Permeability ; Rats ; Ribosomal Protein S6/metabolism* ; Seminiferous Epithelium/metabolism* ; Sertoli Cells/metabolism* ; Signal Transduction/physiology*

Chronic cerebral hypoperfusion (CCH), which is associated with onset of vascular dementia, causes cognitive impairment and neuropathological alterations in the brain. In the present study, we examined the neuroprotective effect of duloxetine (DXT), a potent and balanced serotonin/norepinephrine reuptake inhibitor, on CCH-induced neuronal damage in the hippocampal CA1 region using a rat model of permanent bilateral common carotid arteries occlusion. We found that treatment with 20 mg/kg DXT could attenuate the neuronal damage, the reduction of phosphorylations of mTOR and p70S6K as well as the elevations of TNF-α and IL-1β levels in the hippocampal CA1 region at 28 days following CCH. These results indicate that DXT displays the neuroprotective effect against CCH-induced hippocampal neuronal death, and that neuroprotective effect of DXT may be closely related with the attenuations of CCH-induced decrease of mTOR/p70S6K signaling pathway as well as CCH-induced neuroinflammatory process.
Brain ; CA1 Region, Hippocampal ; Carotid Artery, Common ; Cognition Disorders ; Dementia, Vascular ; Duloxetine Hydrochloride* ; Models, Animal ; Neurons* ; Neuroprotection ; Neuroprotective Agents* ; Phosphorylation ; Ribosomal Protein S6 Kinases, 70-kDa

Mast cells integrate innate and adaptive immunity and are implicated in pathophysiological conditions, including allergy, asthma, and anaphylaxis. Cross-linking of the high-affinity IgE receptor (FcεRI) initiates diverse signal transduction pathways and induces release of proinflammatory mediators by mast cells. In this study, we demonstrated that hyperactivation of mechanistic target of rapamycin (mTOR) signaling using the mTOR activator MHY1485 suppresses FcεRI-mediated mast cell degranulation and cytokine secretion. MHY1485 treatment increased ribosomal protein S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) phosphorylation, which are downstream targets of mTOR complex 1 (mTORC1), but decreased phosphorylation of Akt on mTOR complex 2 (mTORC2) target site serine 473. In addition, this activator decreased β-hexosaminidase, IL-6, and tumor necrosis factor α (TNF-α) release in murine bone marrow-derived mast cells (BMMCs) after FcεRI stimulation. Furthermore, MHY1485-treated BMMCs showed significantly decreased proliferation when cultured with IL-3. These findings suggested hyperactivation of mTORC1 as a therapeutic strategy for mast cell-related diseases.
Adaptive Immunity ; Anaphylaxis ; Asthma ; Cell Degranulation ; Cell Proliferation ; Hypersensitivity ; Immunoglobulin E ; Interleukin-3 ; Interleukin-6 ; Mast Cells* ; Peptide Initiation Factors ; Phosphorylation ; Ribosomal Protein S6 Kinases ; Serine ; Signal Transduction ; Sirolimus ; Tumor Necrosis Factor-alpha