The present study aimed to investigate the effects of eccentric treadmill exercise on adaptive hypertrophy of skeletal muscle in rats. Thirty-two 3-month-old Sprague Dawley (SD) rats were selected and randomly assigned to one of the four groups based on their body weights: 2-week quiet control group (2C), 2-week downhill running exercise group (2E), 4-week quiet control group (4C), and 4-week downhill running exercise group (4E). The downhill running protocol for rats in the exercise groups involved slope of -16°, running speed of 16 m/min, training duration of 90 min, and 5 training sessions per week. Twenty-four hours after the final session of training, all the four groups of rats underwent an exhaustion treadmill exercise. After resting for 48 h, all the rats were euthanized and their gastrocnemius muscles were harvested for analysis. HE staining was used to measure the cross-sectional area (CSA) and diameter of muscle fibers. Transmission electron microscope was used to observe the ultrastructural changes in muscle fibers. Purithromycin surface labeling translation method was used to measure protein synthesis rate. Immunofluorescence double labeling was used to detect the colocalization levels of lysosomal-associated membrane protein 2 (Lamp2)-leucyl-tRNA synthetase (LARS) and Lamp2-mammalian target of rapamycin (mTOR). Western blot was used to measure the protein expression levels of myosin heavy chain (MHC) IIb and LARS, as well as the phosphorylation levels of mTOR, p70 ribosomal protein S6 kinase (p70S6K), and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1). The results showed that, compared with the 2C group rats, the 2E group rats showed significant increases in wet weight of gastrocnemius muscle, wet weight/body weight ratio, running distance, running time, pre- and post-exercise blood lactate levels, myofibrillar protein content, colocalization levels of Lamp2-LARS and Lamp2-mTOR, and LARS protein expression. Besides these above changes, compared with the 4C group, the 4E group further exhibited significantly increased fiber CSA, fiber diameter, protein synthesis rate, and phosphorylation levels of mTOR, p70S6K, and 4E-BP1. Compared with the quiet control groups, the exercise groups exhibited ultrastructural damage of rat gastrocnemius muscle, which was more pronounced in the 4E group. These findings suggest that eccentric treadmill exercise may promote mTOR translocation to lysosomal membrane, activating mTOR signaling via up-regulating LARS expression. This, in turn, increases protein synthesis rate through the mTOR-p70S6K-4E-BP1 signaling pathway, promoting protein deposition and inducing adaptive skeletal muscle hypertrophy. Although the ultrastructural changes of skeletal muscle are more pronounced, the relatively long training cycles during short-term exercise periods have a more significant effect on promoting gastrocnemius muscle protein synthesis and adaptive hypertrophy.
Animals ; Rats, Sprague-Dawley ; Physical Conditioning, Animal/physiology* ; Rats ; Muscle, Skeletal/metabolism* ; TOR Serine-Threonine Kinases/metabolism* ; Male ; Hypertrophy ; Adaptation, Physiological/physiology* ; Adaptor Proteins, Signal Transducing/metabolism* ; Ribosomal Protein S6 Kinases, 70-kDa/metabolism* ; Intracellular Signaling Peptides and Proteins

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 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: 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

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

OBJECTIVE: To investigate the effect of rosuvastatin on homocysteine (Hcy) induced mousevascular smooth muscle cells(VSMCs) dedifferentiation and endoplasmic reticulum stress(ERS). METHODS: VSMCs were co-cultured with Hcy and different concentration of rosuvastatin (0.1, 1.0 and 10 μmol/L). Cytoskeleton remodeling, VSMCs phenotype markers (smooth muscle actin-α, calponin and osteopontin) and ERS marker mRNAs (Herpud1, XBP1s and GRP78) were detected at predicted time. Tunicamycin was used to induce, respectively 4-phenylbutyrate(4-PBA) inhibition, ERS in VSMCs and cellular migration, proliferation and expression of phenotype proteins were analyzed. Mammalian target of rapamycin(mTOR)-P70S6 kinase (P70S6K) signaling agonist phosphatidic acid and inhibitor rapamycin were used in Rsv treated VSMCs. And then mTOR signaling and ERS associated mRNAs were detected. RESULTS: Compared with Hcy group, Hcy+ Rsv group (1.0 and 10 μmol/L) showed enhanced α-SMA and calponin expression (<0.01), suppressed ERS mRNA levels (<0.01) and promoted polarity of cytoskeleton. Compared with Hcy group, Hcy+Rsv group and Hcy+4-PBA group showed suppressed proliferation, migration and enhanced contractile protein expression (<0.01); while tunicamycin could reverse the effect of Rsv on Hcy treated cells. Furthermore, alleviated mTOR-P70S6K phosphorylation and ERS (<0.01)were observed in Hcy+Rsv group and Hcy+rapamycin group, compared with Hcy group; while phosphatidic acid inhibited the effect of Rsv on mTOR signaling activation and ERS mRNA levels (<0.01). CONCLUSIONS Rosuvastatin could inhibit Hcy induced VSMCs dedifferentiation suppressing ERS, which might be regulated by mTOR-P70S6K signaling.
Actins ; metabolism ; Animals ; Calcium-Binding Proteins ; metabolism ; Cell Dedifferentiation ; drug effects ; Cells, Cultured ; Endoplasmic Reticulum Stress ; drug effects ; Heat-Shock Proteins ; metabolism ; Homocysteine ; Membrane Proteins ; metabolism ; Mice ; Microfilament Proteins ; metabolism ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; drug effects ; Ribosomal Protein S6 Kinases, 70-kDa ; metabolism ; Rosuvastatin Calcium ; pharmacology ; TOR Serine-Threonine Kinases ; metabolism ; X-Box Binding Protein 1 ; metabolism

Impaired angiogenesis is one of the crucial factors that impede the wound healing process in diabetic foot ulcers (DFUs). In this study, we found that 20(S)-protopanaxadiol (PPD), an aglycone of ginsenosides in Panax notoginseng, stimulated angiogenesis and benefited wound healing in genetically diabetic mice. In HUVECs, PPD promoted cell proliferation, tube formation and VEGF secretion accompanied by increased nuclear translocalization of HIF-1α, which led to elevated VEGF mRNA expression. PPD activated both PI3K/Akt/mTOR and Raf/MEK/ERK signaling pathways in HUVECs, which were abrogated by LY294002 and PD98059. Furthermore, these two pathways had crosstalk through p70S6K, as LY294002, PD98059 and p70S6K siRNA abolished the angiogenic responses of PPD. In the excisional wound splinting model established in db/db diabetic mice, PPD (0.6, 6 and 60 mg ml−1) accelerated wound closure, which was reflected by a significantly reduced wound area and epithelial gaps, as well as elevated VEGF expression and capillary formation. In addition, PPD activated PI3K/Akt/ERK signaling pathways, as well as enhanced p70S6K activity and HIF-1α synthesis in the wounds. Overall, our results revealed that PPD stimulated angiogenesis via HIF-1α-mediated VEGF expression by activating p70S6K through PI3K/Akt/mTOR and Raf/MEK/ERK signaling cascades, which suggests that the compound has potential use in wound healing therapy in patients suffering from DFUs.
Animals ; Capillaries ; Cell Proliferation ; Diabetic Foot ; Ginsenosides ; Humans ; Mice* ; Panax notoginseng ; Phosphotransferases* ; Ribosomal Protein S6 Kinases, 70-kDa ; RNA, Messenger ; RNA, Small Interfering ; Splints ; Ulcer ; Vascular Endothelial Growth Factor A* ; Wound Healing* ; Wounds and Injuries*

PURPOSE: The purpose of this study was to observe the effects of metformin on human esophageal cancer cell and to investigate its possible mechanisms. MATERIALS AND METHODS: Cell viability was detected by using a Cell Counting Kit-8, while cell cycle and apoptosis were assessed by flow cytometry and western blot was used to measure the expression of the related proteins. RNAi was used to knockout pyruvate kinase muscle isozyme 2 (PKM2). An Eca109 tumor model was established to evaluate the antitumor effect in vivo. Immunohistochemistry was determined based on the expression of PKM2 and Bim in tumor tissues. Tunnel was used to assess tumor cell apoptosis. RESULTS: Esophageal cancer cells viability was reduced after metformin treatment. The cell cycle was arrested in the G0/G1 phase, apoptosis was induced, caspase 3 was activated, caspase 9 was downregulated, and the pro-apoptotic protein Bim increased. Further study revealed that metformin could suppress the expression of insulin-like growth factor 1 receptor and its downstream proteins, phosphoinositide 3-kinase (PI3K), protein kinase B (AKT/PKB), phosphorylation of AKT (pAKT), mammalian target of rapamycin (mTOR), p70S6K, and PKM2. Insulin-like growth factor 1 partly reversed metfromin-induced apoptosis and attenuated the repression effect of metfomin to PI3K, pAKT, and PKM2. Knockout PKM2 resulted in the activation of caspase 3, down-regulation of caspase 9, and increased expression of Bim. In the Eca109 xenograft model, metformin significantly reduced tumor growth. Furthermore, we found that metformin treatment increased the rate of apoptosis, down-regulation of PKM2, and up-regulation of Bim in tumor tissues. CONCLUSION: Metformin restrained esophageal cancer cell proliferation partly by suppressing the PI3K/AKT/mTOR pathway.
Apoptosis ; Blotting, Western ; Caspase 3 ; Caspase 9 ; Cell Count ; Cell Cycle ; Cell Proliferation ; Cell Survival ; Down-Regulation ; Esophageal Neoplasms* ; Flow Cytometry ; Heterografts ; Humans ; Immunohistochemistry ; In Vitro Techniques* ; Metformin* ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; Pyruvate Kinase ; Repression, Psychology ; Ribosomal Protein S6 Kinases, 70-kDa ; RNA Interference ; Sirolimus ; Up-Regulation

PURPOSE: The purpose of this study was to observe the effects of metformin on human esophageal cancer cell and to investigate its possible mechanisms. MATERIALS AND METHODS: Cell viability was detected by using a Cell Counting Kit-8, while cell cycle and apoptosis were assessed by flow cytometry and western blot was used to measure the expression of the related proteins. RNAi was used to knockout pyruvate kinase muscle isozyme 2 (PKM2). An Eca109 tumor model was established to evaluate the antitumor effect in vivo. Immunohistochemistry was determined based on the expression of PKM2 and Bim in tumor tissues. Tunnel was used to assess tumor cell apoptosis. RESULTS: Esophageal cancer cells viability was reduced after metformin treatment. The cell cycle was arrested in the G0/G1 phase, apoptosis was induced, caspase 3 was activated, caspase 9 was downregulated, and the pro-apoptotic protein Bim increased. Further study revealed that metformin could suppress the expression of insulin-like growth factor 1 receptor and its downstream proteins, phosphoinositide 3-kinase (PI3K), protein kinase B (AKT/PKB), phosphorylation of AKT (pAKT), mammalian target of rapamycin (mTOR), p70S6K, and PKM2. Insulin-like growth factor 1 partly reversed metfromin-induced apoptosis and attenuated the repression effect of metfomin to PI3K, pAKT, and PKM2. Knockout PKM2 resulted in the activation of caspase 3, down-regulation of caspase 9, and increased expression of Bim. In the Eca109 xenograft model, metformin significantly reduced tumor growth. Furthermore, we found that metformin treatment increased the rate of apoptosis, down-regulation of PKM2, and up-regulation of Bim in tumor tissues. CONCLUSION: Metformin restrained esophageal cancer cell proliferation partly by suppressing the PI3K/AKT/mTOR pathway.
Apoptosis ; Blotting, Western ; Caspase 3 ; Caspase 9 ; Cell Count ; Cell Cycle ; Cell Proliferation ; Cell Survival ; Down-Regulation ; Esophageal Neoplasms* ; Flow Cytometry ; Heterografts ; Humans ; Immunohistochemistry ; In Vitro Techniques* ; Metformin* ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; Pyruvate Kinase ; Repression, Psychology ; Ribosomal Protein S6 Kinases, 70-kDa ; RNA Interference ; Sirolimus ; Up-Regulation