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

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*

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

BACKGROUNDTriggering receptor expressed on myeloid cell-1 (TREM-1) may play a vital role in mammalian target of rapamycin (mTOR) modulation of CD8+ T-cell differentiation through the transcription factors T-box expressed in T-cells and eomesodermin during the immune response to invasive pulmonary aspergillosis (IPA). This study aimed to investigate whether the mTOR signaling pathway modulates the proliferation and differentiation of CD8+ T-cells during the immune response to IPA and the role TREM-1 plays in this process.

METHODSCyclophosphamide (CTX) was injected intraperitoneally, and Aspergillus fumigatus spore suspension was inoculated intranasally to establish the immunosuppressed IPA mouse model. After inoculation, rapamycin (2 mg.kg-1.d-1) or interleukin (IL)-12 (5 μg/kg every other day) was given for 7 days. The number of CD8+ effector memory T-cells (Tem), expression of interferon (IFN)-γ, mTOR, and ribosomal protein S6 kinase (S6K), and the levels of IL-6, IL-10, galactomannan (GM), and soluble TREM-1 (sTREM-1) were measured.

RESULTSViable A. fumigatus was cultured from the lung tissue of the inoculated mice. Histological examination indicated greater inflammation, hemorrhage, and lung tissue injury in both IPA and CTX + IPA mice groups. The expression of mTOR and S6K was significantly increased in the CTX + IPA + IL-12 group compared with the control, IPA (P = 0.01; P= 0.001), and CTX + IPA (P = 0.034; P= 0.032) groups, but significantly decreased in the CTX + IPA + RAPA group (P < 0.001). Compared with the CTX + IPA group, the proportion of Tem, expression of IFN-γ, and the level of sTREM-1 were significantly higher after IL-12 treatment (P = 0.024, P= 0.032, and P= 0.017, respectively), and the opposite results were observed when the mTOR pathway was blocked by rapamycin (P < 0.001). Compared with the CTX + IPA and CTX + IPA + RAPA groups, IL-12 treatment increased IL-6 and downregulated IL-10 as well as GM, which strengthened the immune response to the IPA infection.

CONCLUSIONSmTOR modulates CD8+ T-cell differentiation during the immune response to IPA. TREM-1 may play a vital role in signal transduction between mTOR and the downstream immune response.

Animals ; CD8-Positive T-Lymphocytes ; cytology ; metabolism ; Cell Differentiation ; genetics ; physiology ; Female ; Interferon-gamma ; metabolism ; Invasive Pulmonary Aspergillosis ; metabolism ; Lymphocyte Activation ; genetics ; physiology ; Mice ; Mice, Inbred BALB C ; Myeloid Cells ; cytology ; metabolism ; Ribosomal Protein S6 Kinases ; metabolism ; TOR Serine-Threonine Kinases ; genetics ; metabolism ; Tissue Culture Techniques

OBJECTIVETo investigate the effect of rapamycin on the proliferation of rat valvular interstitial cells in primary culture.

METHODSThe interstitial cells isolated from rat aortic valves were cultured and treated with rapamycin, and the cell growth and cell cycle changes were analyzed using MTT assay and flow cytometry, respectively. RT-PCR was used to detect mRNA expression levels of S6 and P70S6K in cells, and the protein expressions level of S6, P70S6K, P-S6, and P-P70S6K were detected using Western blotting.

RESULTSRat aortic valvular interstitial cells was isolated successfully. The rapamycin-treated cells showed a suppressed proliferative activity (P<0.05), but the cell cycle distribution remained unaffected. Rapamycin treatment resulted in significantly decreased S6 and P70S6K protein phosphorylation level in the cells (P<0.05).

CONCLUSIONThe mechanism by which rapamycin inhibits the proliferation of valvular interstitial cells probably involves suppression of mTOR to lower S6 and P70S6K phosphorylation level but not direct regulation of the cell cycle.

Animals ; Blotting, Western ; Cell Cycle ; Cell Proliferation ; drug effects ; Cells, Cultured ; Heart Valves ; cytology ; Phosphorylation ; Rats ; Ribosomal Protein S6 Kinases, 70-kDa ; metabolism ; Sirolimus ; pharmacology

BACKGROUNDThe phosphorylation of p70S6 kinase (p70S6K) represents an important target for sensitive detection on pharmacodynamic effects of sirolimus, but the methods of assessing p70S6K phosphorylation are still unclear. The aim of this study was to investigate p70S6K phosphorylation located down-stream of the mammalian target of rapamycin (mTOR) pathway in peripheral blood mononuclear cells (PBMCs) of liver transplant patients through different methods.

METHODSSeventy-five liver transplant recipients from Beijing Chaoyang Hospital of the Capital Medical University were analyzed in this study. Patients were divided into three groups, patient treated with sirolimus (n = 22), patient treated with tacrolimus (n = 30), patient treated with cyclosporine (n = 23). The p70S6K phosphorylation of PBMCs in patients and healthy control (HC, n = 12) were analyzed by phospho-flow cytometry and Western blotting. A correlation analysis of data from phospho-flow cytometry and Western blotting was performed. Intra-assay variability of p70S6K phosphorylation in HC and different patients were measured.

RESULTSIntra-assay variability of p70S6K phosphorylation in phospho-flow cytometry was from 4.1% to 8.4% and in Western blotting was from 8.2% to 18%. The p70S6K phosphorylation in patients receiving a sirolimus (19.5 ± 7.7) was significantly lower than in HC (50.1 ± 11.3, P < 0.001), tacrolimus (37.7 ± 15.7, P < 0.001) or cyclosporine treated patients (41.7 ± 11.7, P < 0.001). The p70S6K phosphorylation in HC (50.1 ± 11.3) was significantly higher than in tacrolimus (37.7 ± 15.7, P < 0.01) or cyclosporine-treated patients (41.7 ± 11.7, P < 0.01). There was correlation between data from phospho-flow cytometry and data from Western blotting (r = 0.88, P < 0.001).

CONCLUSIONSThe degree of mTOR inhibition by assessing p70S6K phosphorylation was established by phospho-flow cytometry and Western blotting. Assessment of p70S6K phosphorylation may play an adjunct role to on pharmacodynamically guide and individualize sirolimus based on immunosuppression.

Adolescent ; Adult ; Aged ; Blotting, Western ; Cyclosporine ; pharmacokinetics ; therapeutic use ; Female ; Flow Cytometry ; Humans ; Immunosuppressive Agents ; pharmacokinetics ; therapeutic use ; Leukocytes, Mononuclear ; enzymology ; Liver Transplantation ; Male ; Middle Aged ; Phosphorylation ; Ribosomal Protein S6 Kinases, 70-kDa ; metabolism ; Sirolimus ; pharmacokinetics ; therapeutic use ; Tacrolimus ; pharmacokinetics ; therapeutic use ; Young Adult

p70 ribosomal protein S6 kinase (p70S6K), an important member of AGC family, is a kind of multifunctional Ser/Thr kinases, which plays an important role in mTOR signaling cascade. The p70 ribosomal protein S6 kinase is closely associated with diverse cellular processes such as protein synthesis, mRNA processing, glucose homeostasis, cell growth and apoptosis. Recent studies have highlighted the important role of S6K in cancer, which arose interests of scientific researchers for the design and discovery of anti-cancer agents. Herein, the mechanisms of S6K and available inhibitors are reviewed.
Antineoplastic Agents ; Humans ; Protein Kinase Inhibitors ; chemistry ; Ribosomal Protein S6 Kinases, 70-kDa ; antagonists & inhibitors ; metabolism ; Signal Transduction ; TOR Serine-Threonine Kinases