Protein Mammalian Target Of Rapamycin Research Articles

Determination of five mTOR inhibitors in human plasma for hepatocellular carcinoma treatment using QuEChERS-UHPLC-MS/MS

A fast and reliable QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method for pre-processing combined with Ultra - high performance liquid chromatography - tandem mass spectrometry (UHPLC-MS/MS) was established for the analysis of five mammalian rapamycin target protein (mTOR) inhibitors (vistusertib, AZD8055, pictilisib, everolimus, temsirolimus)in human plasma. Extraction was achieved by addition of acetonitrile to the sample followed by anhydrous magnesium sulfate and 30 mg C18 for salting out and purification, respectively. MTOR inhibitors were detected using selective response monitoring (SRM) under positive ion electrospray mode. Vistusertib, AZD8055 and pictilisib showed good linearity with a range of 1–80 ng/ml, Additionally, the concentration of everolimus and temsirolimus was 2.5–200 ng/ml and10–800 ng/ml, respectively. The linear correlation coefficient (R2) of each analysis was ≥ 0.9950. The limit of detection (LOD) and Limit of Quantitation (LOQ) were 0.015–0.75 ng/ml and 1–10 ng/ml, respectively. This method showed a high accuracy with high recovery rate and excellent stability. This method is fast, accurate and reliable, suitable for quantitative detection of mTOR inhibitors in human plasma.

Brucea javanica Oil Emulsion Promotes Autophagy in Ovarian Cancer Cells Through the miR-8485/LAMTOR3/mTOR/ATG13 Signaling Axis.

Background: Ovarian cancer is a common malignant tumor of the female reproductive tract, with the highest mortality rate. At present, no effective approaches to improve the survival rate exist. B. javanica Oil Emulsion (BJOE), an extract from B. javanica (L.) Merr. [Simaroubaceae], exhibits antitumor effects and can increase the sensitivity of radiotherapy and chemotherapy in many types of cancers. MiR-8485, a discovered miRNA, has been shown to be involved in the occurrence and development of tumors. The purpose of this study was to investigate the effect of BJOE on the regulation of mammalian rapamycin target protein (mTOR) autophagy signal pathway and related autophagy factors on ovarian cancer cells through miR-8485. Methods: The main chemical constituents of BJOE were determined by UHPLC-MS/MS. Detection of miR-8485 expression in ovarian cancer cells treated with BJOE by quantitative reverse transcription polymerase chain reaction (qRT-PCR). CCK8 experiment and flow cytometry were used to observe the effects of BJOE and overexpression of miR-8485 on cell proliferation and apoptosis. Then, monodansylcadaverine (MDC) fluorescence staining was used to observe the changes of autophagy vesicles before and after the effect of BJOE and overexpressed miR-8485 on cancer cells. Next, the binding sites between miR8485 and mammalian rapamycin target protein activator 3 (LAMTOR3) were detected by double luciferase reporter assay. Furthermore, qRT-PCR and Western blot experiments were used to explore the changes of autophagy-related factors LAMTOR3, mTOR and autophagy-related 13 (ATG13), and microtubule associated protein 1 light chain 3 beta (LC3-Ⅱ) after BJOE and overexpression of miR-8485, in addition to autophagy inhibitor (3-MA) for rescue experiment verification. Results: The qRT-PCR results showed that the expression of miR-8485 increased after BJOE treatment in the SKOV3 cell. The CCK8 assay and flow cytometry analysis revealed that both BJOE and miR-8485 overexpression inhibited the proliferation and promoted the apoptosis of the SKOV3 cell. MDC fluorescence staining showed that BJOE and miR-8485 overexpression led to a significant increase in autophagy vesicles in the SKOV3 cell. Double luciferase reporter assay confirmed the existence of binding sites between miR8485 and LAMTOR3. The results of qRT-PCR and Western blot showed that BJOE and overexpressed miR-8485 downregulated the expression of LAMTOR3 and mTOR and up-regulated the expression of ATG13 and LC3-Ⅱ. Conclusion: 1) MiR-8485 may be the key factor of BJOE in promoting autophagy and apoptosis and inhibiting cell proliferation of ovarian cancer cells; 2) BJOE may play an antitumor role by regulating LAMTOR3/mTOR/ATG13 signaling axis through miR-8485 to promote autophagy in ovarian cancer cells.

Acupuncture regulates PI3K/Akt/mTOR signaling and up-regulates cell autophagy to protect sy-novial tissue of adjuvant arthritis rats

To observe the effect of acupuncture of "Yinlingquan"(SP9) and "Sanyinjiao"(SP6) on expression of phosphatidylinositol-3 kinase/protein kinase B/mammalian target protein of rapamycin (PI3K/Akt/mTOR) signaling in adjuvant arthritis (AA) rats, so as to explore its mechanism underlying improvement of AA. Forty-eight male Wistar rats were randomly divided into normal control, AA model, acupuncture and medication (tripterygium wilfordii) groups, with 12 rats in each group. The AA model was established by putting the rats in a windy, cold and wet environment for 12 h, once every day for 21 days and injection of Freund's complete adjuvant (CFA) into the sole of the right hindlimb on the 21st day. Manual acupuncture stimulation was applied at SP9 and SP6 for 30 min/time, once a day for 21 days. Rats of the medication group received gavage of tripterygium wilfordii tablets solution (8 mg/kg), once a day for 21 days, and those of the normal control group and model group received gavage of the same amount of normal saline, once a day for 21 days. The degree of joint swelling and arthritis index (AI) were detected 1 day before modeling, 3 days after modeling, and 21 days after the treatment. Twenty-four hours after the last treatment, the contents of serum cytokines interleukin (IL)-17, IL-6 and tumor necrosis factor (TNF)-α were detected by enzyme-linked immunosorbent assay (ELISA); and changes of synovial ultrastructure were observed under electron microscope. Western blot was used to detect the expression levels of PI3K, Akt, phosphorylated protein kinase B (p-Akt), phosphorylated mammalian rapamycin target protein (p-mTOR), mTOR, microtubule associated protein 1 light chain 3B (LC3-Ⅱ) and mammalian atg6 homologous protein (Beclin-1) in the synovial membrane tissue. Compared with the normal control group, the joint swel-ling degree and AI, contents of serum TNF-α, IL-6 and IL-17, and the expression levels of PI3K, Akt, p-Akt, mTOR and p-mTOR in the synovium were increased in the model group (P<0.05), while the expression levels of LC3-Ⅱ and Beclin-1 proteins in the synovium were significantly decreased (P<0.05). Compared with the model group, the joint swelling degree and AI, contents of serum TNF-α, IL-6 and IL-17, and the expression levels of PI3K, Akt, p-Akt, mTOR and p-mTOR were significantly decreased in the acupuncture and medication groups (P<0.05), while the expresion levels of LC3-Ⅱ and Beclin-1 proteins were significantly increased （P<0.05）. Comparison between the two treatment groups showed that the therapeutic effects of acupuncture were obviously weaker than those of medication in down-regulating TNF-α and IL-6, Akt, p-Akt and mTOR levels (P<0.05) and in up-regulating Beclin-1 expression (P<0.05). Outcomes of electron microscope displayed widened nuclear membrane space, some fractured mitochondrial cristae with vacuoles, expanded rough endoplasmic reticulum, reduction of autophagosomes in the cytoplasm and ruptured synovial cell membrane in the model group, and increase of autophagosomes, deformed organelles in the acupuncture and medication groups. Acupuncture can relieve the inflammatory reactions and joint synovial injury of the affected joint in AA rats which may be associated with its effects in inhibiting PI3K/Akt/mTOR signaling and increasing the auto-phagy level of synovial cells.

Alteration in the mRNA expression profile of the autophagy-related mTOR pathway in schizophrenia patients treated with olanzapine

BackgroundThe mammalian target of rapamycin protein (mTOR) signaling pathway is involved in the pathogenesis of schizophrenia and the mechanism of extrapyramidal adverse reactions to antipsychotic drugs, which might be mediated by an mTOR-dependent autophagy impairment. This study aimed to examine the expression of mTOR pathway genes in patients with schizophrenia treated with olanzapine, which is considered an mTOR inhibitor and autophagy inducer.MethodsThirty-two patients with acute schizophrenia who had been treated with olanzapine for four weeks (average dose 14.24 ± 4.35 mg/d) and 32 healthy volunteers were recruited. Before and after olanzapine treatment, the Positive and Negative Syndrome Scale (PANSS) was used to evaluate the symptoms of patients with schizophrenia, and the mRNA expression levels of mTOR pathway-related genes, including MTOR, RICTOR, RAPTOR, and DEPTOR, were detected in fasting venous blood samples from all subjects using real-time quantitative PCR.ResultsThe MTOR and RICTOR mRNA expression levels in patients with acute schizophrenia were significantly decreased compared with those of healthy controls and further significantly decreased after four weeks of olanzapine treatment. The DEPTOR mRNA expression levels in patients with acute schizophrenia were not significantly different from those of healthy controls but were significantly increased after treatment. The expression levels of the RAPTOR mRNA were not significantly different among the three groups. The pairwise correlations of MTOR, DEPTOR, RAPTOR, and RICTOR mRNA expression levels in patients with acute schizophrenia and healthy controls were significant. After olanzapine treatment, the correlations between the expression levels of the DEPTOR and MTOR mRNAs and between the DEPTOR and RICTOR mRNAs disappeared.ConclusionsAbnormalities in the mTOR pathway, especially DEPTOR and mTORC2, might play important roles in the autophagy mechanism underlying the pathophysiology of schizophrenia and effects of olanzapine treatment.

Hydrogen-saturated saline mediated neuroprotection through autophagy via PI3K/AKT/mTOR pathway in early and medium stages of rotenone-induced Parkinson’s disease rats

Some cardiovascular symptoms in the early stage of Parkinson’s disease (PD) were related to degeneration of the rostral ventrolateral medulla (RVLM) catecholaminergic neurons. To date, little is known about the effects of hydrogen water on early stage of PD. Here, protective actions of hydrogen-saturated saline (HS) on rotenone-induced PD rats, as well as its underlying mechanisms were investigated. HS was used to treat PD rats at three general stages; early, medium and late, which were represented by rotenone induced rats for 0, 7 and 14 days. HS treatment significantly alleviated the cardiovascular and motor symptoms in rotenone-induced PD rats, improved the survival number of RVLM catecholaminergic neurons and nigral dopamine neurons only in early and medium stages of PD rats. Decreased levels of reactive oxygen species (ROS) and alpha-synuclein (α-Syn), transformation of microtubule associated protein 1 light chain 3 (LC3)-I/II and degradation of sequestosome 1 (p62) were detected, as well as increased expression level of autophagy related protein 5 (ATG5) and B-cell lymphoma-2 interacting protein 1 (Beclin-1) in the RVLM and substantia nigra (SN) after HS treatment in early and medium stages of PD rats. In addition, phosphorylation levels of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt) and mammalian rapamycin target protein (mTOR) decreased after HS treatment in early and medium stages of PD rats. The results suggested that HS treatment exerted beneficial effects in early and medium stages before motor impairments emerged but not in the late stage of rotenone-induced PD rats. It exerted neuroprotection with RVLM catecholaminergic neurons and nigral dopamine neurons, mediated in part by decreasing levels of ROS and α-Syn through increasing autophagy machinery which were partly via inhibiting PI3K-Akt-mTOR pathway.

In silico and In vitro Perspective to Elucidate the Anticancer Effect of Natural Compound Hesperetin in Inhibiting Mammalian Target of Rapamycin Pathway

In this study we intended to perform in silico and in vitro analysis of naturally derived flavonoid, hesperetin as a promising mammalian target of rapamycin inhibitor. This study features in silico screening of natural compound hesperetin against mammalian target of rapamycin FK506 binding proteins 12 domain by using Schrodinger glide based virtual screening and further molecular dynamics simulation was done to infer the stability of complex formation. Mammalian target of rapamycin co-crystallized with P2X (a known inhibitor of mTOR) (Protein Data Bank ID: 4JT5) was used for reference guided docking protocol. Then in vitro analysis applying hesperetin for cell viability followed by protein expressions in ovarian PA-1 and MCF-7 breast cancer cell lines were performed dose dependently. in silico molecular docking and dynamic studies with mammalian target of rapamycin protein revealed that hesperetin was found to be a potent compound that could inhibit the FK506 binding proteins 12 domain of mammalian target of rapamycin. The in vitro studies with hesperetin on PA-1 and MCF-7 breast cancer cell lines, found that hesperetin in a dose dependent manner significantly reduced the phosphorylation of mammalian target of rapamycin on Serine 2448 a marker for mammalian target of rapamycin complex 1 activity, as well as phosphorylation of eukaryotic translation initiation factor 4E binding protein 1 and Ribosomal protein S6 kinase beta-1 which are downstream molecules of mammalian target of rapamycin was also significantly inhibited. To the best of our knowledge this is the first study that brings up the scientific evidence for the efficacy of hesperetin in inhibiting mammalian target of rapamycin with a similar docking score to the native ligand and also in a dose dependent manner in PA-1 human ovarian cancer cells and MCF-7 breast cancer cells, as an anticancer agent with further studies in near future.

Topical administration of rapamycin promotes retinal ganglion cell survival and reduces intraocular pressure in a rat glaucoma model

Glaucoma is a progressive optic neuropathy that has become the most common cause of irreversible blindness worldwide. Studies have shown that the protein mammalian target of rapamycin (mTOR) is a serine/threonine kinase that plays a central role in regulating numerous functions, such as growth, proliferation, cytoskeletal organization, metabolism, and autophagy. Clinical trials have shown that Rho-associated protein kinase (ROCK) inhibitors reduced intraocular pressure (IOP) in patients with glaucoma and ocular hypertension (OHT). In this study, we explored whether rapamycin (RAPA) eye drops can reduce IOP and protect retinal ganglion cells (RGCs). Our results indicated that in rats treated with RAPA, the drug was detected in the aqueous humor (AH), and the IOP was reduced. This may be related to the inhibition of RhoA protein activation by RAPA and regulation of the actin cytoskeleton in trabecular meshwork (TM) cells. In addition, the retinal thickness and the survival rate of RGCs were significantly reduced in the OHT group compared with the control group. These changes in the OHT group were significantly improved after treatment with RAPA. This may be because RAPA inhibited the activation of glial cells and the release of proinflammatory factors, thereby attenuating further damage to the retina and RGCs. Taken together, the results of this study demonstrated that RAPA not only reduced IOP but also protected RGCs, suggesting that RAPA is likely to be an effective strategy for the treatment of glaucoma.

Effects of propofol on TGF-β1 induced activation of hepatic stellate cells and its mechanism

Objective: To observe the effects of propofol on the activation of hepatic stellate cell line HSC2-T6 induced by transforming growth factor-beta 1 (TGF-β1) and explore its possible mechanism.Methods: The cells were divided into control group, TGF-β1 group, propofol group, TGF-β1 + propofol group, rapamycin group, TGF-β1 + propofol + rapamycin group. Cells were treated with rapamycin (5 μmol/L) for 1 hour, propofol (100 μmol/L) for 1 hour, then TGF-β1 (5 ng/ml) was added to co-culture for 24 hours. Cell proliferation was measured by MTT assay. The concentrations of hyaluronic acid (HA), collagen IV (IV-C) and laminin (LN) in the supernatant of cell culture medium were measured by ELISA. The ultrastructure of cells was observed by transmission electron microscopy. The expressions of alpha-smooth muscle actin (α-SMA), mammalian rapamycin target protein (mTOR), phosphorylated mTOR (p-mTOR) and the autophagy related gene Beclin 1, LC3 and p62 were measured by Western blot. Results: Compared with control group, cell proliferation, the expression of α-SMA, the concentrations of HA, IV-C and LN in culture supernatant, the number of autophages, the expressions of Beclin-1 and LC3-II, the ratio of LC3-II/LC3-I in HSC2-T6 cells were increased significantly, while the expression of p-mTOR, the ratio of p-mTOR/mTOR and the expression of p62 protein were decreased significantly in TGF-β1 group (All P＜0.05). Compared with TGF-β1 group, cell proliferation, the expression of α-SMA, the concentrations of HA, IV-C and LN in culture supernatant, the number of autophages, the expressions of Beclin-1 and LC3-II, the ratio of LC3-II/LC3-I in HSC2-T6 cells in TGF-β1 group were decreased significantly, and the expression of p-mTOR, the ratio of p-mTOR/mTOR and expression of p62 protein were increased significantly in TGF-β1 + propofol group (All P＜0.05). Conclusion: Propofol inhibits the activation of hepatic stellate cells induced by TGF-beta 1, and its mechanism involves the mTOR-autophagy pathway.

Effect and mechanism of silent information regulator 6 on gluconeogenesis in chronic-on-acute liver failure

Objective To investigate the effect and mechanism of silent information regulator 6 (SIRT6) and gluconeogenesis-dependent rate-limiting enzymes in hepatocytes in oxidative stress injury rats and chronic-on-acute (sub-acute) liver failure (ACLF) patients. Methods From August 2016 to May 2018, 10 patients with ACLF from Beijing Youan Hospital Affiliated to Capital Medical University were included in the ACLF group, and 10 normal donors were included in the normal control group. Level of fasting blood glucose, total bilirubin, albumin, and alanine aminotransferase (ALT) were studied. Sprague Dawley rat hepatocytes were isolated and divided into control group (without any intervention), model group (H2O2 intervention for 6 h), mammalian rapamycin target protein (mTOR) activation group (mTOR activation was added to the model group), mTOR inhibition group (mTOR inhibitor was added on the basis of the model group). Protein electrophoresis and polymerase chain reaction was used to detect the relative expression of glucose-6-phosphatase (G6P), phosphoenolpyruvate (PEPCK), SIRT6, and mTOR. Results The ALT and total bilirubin level in ACLF group were significantly higher than those in the normal control group, and the differences were statistically significant (all P<0.05). In ACLF group, level of SIRT6 (0.15±0.07) μg/L and fasting blood glucose (3.19±0.59) mmol/L were significantly lower than those in the normal control group (0.46±0.15) μg/L and (7.07±2.07) mmol/L, the difference was statistically significant (all P<0.05). The relative expression of PEPCK and G6P protein in liver tissue of ACLF group was significantly lower than that of normal control group. The relative expression of SIRT6, PEPCK, and G6P in the model group were lower than those in the control group, and the differences were statistically significant (all P<0.05). When mTOR is activated, the relative expression of PEPCK, G6P, and SIRT6 was higher than those in the model group, and after mTOR inhibition, the relative expression of PEPCK, G6P, and SIRT6 was lower than in the model group. Conclusion ACLF, SIRT6 may inhibit gluconeogenesis, and increased the occurrence of hypoglycemia through activating mTOR signaling pathway. Blocking of SIRT6 levels may slow down the progress of ACLF. Key words: Acute-on-chronic liver failure; Glucose-6-phosphatase; Phosphoenolpyruvate carboxykinase; Silent information regulator 6; Mammalian rapamycin target protein

Naringin inhibits autophagy mediated by PI3K-Akt-mTOR pathway to ameliorate endothelial cell dysfunction induced by high glucose/high fat stress

As a flavonoid, naringin (Nar) has been shown to have multiple pharmacological effects including lowering blood cholesterol, reducing thrombus formation and improving microcirculation. However, effects of Nar on function and autophagy of vascular endothelial cells under high glucose and high fat (HG/HF) stress are largely unclear. This study was designed to investigate such effects of Nar in human umbilical vein endothelial cells (HUVECs) and to determine whether such effects are related to autophagy. Our present results show that 86 μM of Nar inhibits the autophagy levels and protects the cells against the dysfunction induced by HG/HF stress. Moreover, Nar increases the phosphorylation levels of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt) and mammalian rapamycin target protein (mTOR). However, pretreatment with rapamycin (RAPA, 5 μM, autophagy inducer), LY294002(10 μM, PI3K inhibitor) and Akt inhibitor Ⅳ (0.5 μM, Akt inhibitor) partially abrogates the protective effects of Nar, suggesting that the protective effects of Nar are achieved by activating the PI3K-Akt-mTOR pathway to inhibit autophagy. In conclusion, Nar improves the function of HUVECs under HG/HF stress through activating the PI3K-Akt-mTOR pathway to inhibit autophagy. The findings offer an insight into HG/HF stress-induced autophagy and indicate that Nar might have potential to prevent and treat the diabetic angiopathy.

Changes in cecal morphology, cell proliferation, antioxidant enzyme, volatile fatty acids, lipopolysaccharide, and cytokines in piglets during the postweaning period

Weaning stresses often induce markedly structural and functional changes in the small intestine. However, little data are available on the changes of the morphology, function, and health in the cecum of piglets post-weaning. This study was conducted to measure the effects of weaning on the cecum in piglets. Forty piglets were weaned at 21 d and samples were collected at different time points (days 0, 1, 3, 7, and 14) post-weaning. The crypt depth, crypt width, and total epithelial cell numbers increased on days 7 and 14, compared with days 0, 1, and 3 (P < 0.001). The value of Ki67/total epithelial cells was highest on day 0 compared with all other days (P < 0.05). Besides, a higher concentration of malondialdehyde was observed on day 3 (P < 0.001). The activity of CuZn-superoxide dismutase (P < 0.05) enhanced from day 0 to 1 and the activity of catalase increased from day 1 to 3 (P < 0.001). In addition, days 3, 7, and 14 had greater acetic, propanoic, and butyric acid contents than on day 1 (P < 0.001). The pH and monocarboxylate transporter 1 (MCT1) expression increased from day 0 to 1 and from day 3 to 7 and decreased between days 1 and 3 (P < 0.001). The mRNA expression of solute carrier family 9 member A3 (SLC9A3) decreased on day 1 compared to all other postweaning days (P < 0.05). The abundance of toll-like receptor 4 (TLR4) and interferon-γ (IFN-γ) mRNA expression increased (P < 0.05) during the first 24 h after weaning. The concentration of lipopolysaccharide increased from day 3 to 7, then decreased on day 14 (P < 0.05). The mRNA expression of tumor necrosis factor-α (TNF-α) enhanced from day 7 to 14 (P < 0.05). The abundance of phosphorylated mammalian target of rapamycin protein was lower on day 14 than day 0 (P < 0.05). Taken together, these results show that weaning in piglets influences intestinal morphology, function, and health in the cecum.

Role and mechanism of splenic myeloid-derived suppressor cells in sepsis-induced adrenal injury in mice

To investigate the role and mechanism of splenic myeloid-derived suppressor cells (MDSCs) in sepsis-induced adrenal injury (SAI). Thirty male C57 mice aged 6-8 weeks were randomly divided into normal control group (n = 5), sham operation group (Sham group, n = 5), sepsis model group [cecal ligation and perforation (CLP) group, n = 10] and sepsis+splenectomy group (CLPS group, n = 10). The sepsis model of mice was reproduced by CLP method. In Sham group, only the cecum was opened and separated, then closed, without CLP. In CLPS group, the spleen was removed before CLP. In normal control group, no challenge was given. After 24 hours, the rats were sacrificed by anesthesia, and peripheral blood, spleen, bone marrow, and bilateral adrenal glands were harvested. The pathological of adrenal gland was assessed by hematoxylin-eosin (HE) staining under optical microscope. The ratio of MDSCs in peripheral blood, spleen and bone marrow was determined by flow cytometry. The expressions of MDSCs surface antigen CD11b, Gr-1 and interleukins (IL-6, IL-1β) mRNA in adrenal tissue were measured by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). Western Blot was used to detect the expressions of mammalian rapamycin target protein (mTOR) pathway related proteins including total mTOR (T-mTOR), phosphorylation of mTOR (p-mTOR) and caspase-3. The adrenal cortex and medulla of the normal control group and Sham group were intact and the structure was clear under optical microscope, while in the CLP group, the adrenal gland showed edema, cortical hemorrhage and cell edema. Compared with the CLP group, the adrenal tissue injury was significantly reduced in the CLPS group. Compared with the normal control group and Sham group, MDSCs ratio in the peripheral blood was significantly increased and significantly reduced in the spleen in the CLP group, but there was no significant difference in bone marrow, the expression levels of CD11b, Gr-1, IL-6, IL-1β mRNA and caspase-3 protein were increased significantly and p-mTOR protein expression was significantly decreased in adrenal tissue, there was no significant difference in the expression of T-mTOR protein. Compared with the CLP group, in the CLPS group, the MDSCs ratio in the peripheral blood was significantly decreased (0.143±0.011 vs. 0.324±0.023, P < 0.01), the expression levels of CD11b, Gr-1, IL-6 , IL-1β mRNA and caspase-3 protein in adrenal gland were significantly decreased [CD11b mRNA (2-ΔΔCt): 2.90±0.56 vs. 5.74±0.13, Gr-1 mRNA (2-ΔΔCt): 2.71±0.14 vs. 4.59±0.46, IL-6 mRNA (2-ΔΔCt): 2.44±0.64 vs. 5.17±1.04, IL-1β mRNA (2-ΔΔCt): 3.58±0.52 vs. 4.44±0.26, caspase-3 protein (caspase-3/GAPDH): 0.05±0.01 vs. 0.13±0.02, all P < 0.01], the p-mTOR protein expression was significantly increased (p-mTOR/GAPDH: 0.61±0.11 vs. 0.27±0.04, P < 0.01). The spleen is the major source of MDSCs in SAI. Splenectomy can attenuate SAI by reducing mobilization of MDSCs and activating the mTOR signaling pathway.

Proliferation inhibition and apoptosis promotion induced by deletion of TMPO in A549

Objective: To investigate the effect of thymopoietin (TMPO) gene deleted by small interfering RNA (RNAi) on the proliferation and apoptosis of lung cancer cell A549 and its mechanism. Methods: TMPO siRNA was transfected into A549 cells by lipofection. The transfected siRNA control was used as a negative control, and the parent cells were used as blank control. Forty-eight hours later, the expression of TMPO in the transfected cells was detected by real-time fluorescent quantitative polymerase chain reaction (RT-PCR) and Western blot. Cell proliferation was detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay, cell cycle and apoptosis were detected by flow cytometry, the protein levels of proliferating cell nuclear antigen (PCNA), cleaved caspase-3, notch receptor 1 (Notch1) and mammalian rapamycin target protein (mTOR) were detected by Western blot analysis. Results: The results of RT-PCR and Western blot showed that the expression levels of TMPO mRNA in the blank control group, the negative control group and TMPO siRNA transfected group were (1.01±0.11), (0.99±0.10), (0.36±0.04), respectively, the protein levels were (0.27±0.02), (0.29±0.03), (0.08±0.10), respectively. The expression levels of TMPO mRNA and protein in the transfected group were significantly lower than those in the blank control and negative control group (P<0.05). The results of MTT assay showed that the OD values of the blank control group, the negative control group and the transfected group were (0.35±0.04), (0.37±0.04) and (0.34±0.03) at 24 h of transfection, respectively. The OD values at 48 h were (0.47±0.06), (0.46±0.08), (0.37±0.04), the OD values at 72 h were (0.75±0.08), (0.73±0.07), (0.49±0.05), respectively, and the OD values at 96 h were (1.09±0.07), (1.06±0.08), (0.56±0.06). The proliferation abilities of the transfected cells at 48, 72, 96 h were significantly lower than those of the blank control and the negative control group (P<0.05). The results of flow cytometry showed that the proportion of G(0)/G(1) phase cells in blank control group, negative control group and transfection group were (62.55±2.03)%, (61.24±3.15)%, (47.35±2.44)%, respectively. The proportion of cells in S phase were (17.12±1.31)%, (17.70±2.01)%, and (20.81±2.06)%, respectively. The proportion of cells in G(2)/M phase were (20.33±1.43)%, (21.06±1.52)%, (31.84±2.76)%, respectively. The proportion of cells in G(0)/G(1) phase of transfection group was significantly lower than those of blank control and negative control group (P<0.05). The proportion of cells in G(2)/M phase of transfection group was significantly higher than those of blank control and negative control group (P<0.05). The apoptosis ratio of the transfection group was (34.10±2.69)%, significantly higher than (2.96±0.03)% of the blank control and (3.01±0.04)% of the negative control group (P<0.05). Western blot analysis showed that PCNA, Notch1 and mTOR proteins were down-regulated while cleaved caspase-3 protein was up-regulated in A549 cells after deletion of TMPO. Conclusion: The inhibition of TMPO gene expression induced by small interfering RNA can significantly inhibit the proliferation and induce apoptosis of A549 cells, and the mechanism is associated with the inhibition of the activation of Notch1/mTOR signaling pathway.

Effect of Dietary Inulin Supplementation on Growth Performance, Carcass Traits, and Meat Quality in Growing-Finishing Pigs.

Simple SummaryDietary fiber has attracted considerable research interest worldwide. Inulin is a critical soluble dietary fiber. This study investigated the effects of dietary inulin supplementation on the growth performance and meat quality in pigs, which provided novel insights into the application of inulin for the livestock industry.Inulin is one of the commercially feasible dietary fibers that has been implicated in regulating the gut health and metabolism of animals. This experiment was conducted to investigate the effect of dietary inulin supplementation on growth performance and meat quality in growing–finishing pigs. Thirty-six Duroc × Landrace × Yorkshire White growing barrows (22.0 ± 1.0 kg) were randomly allocated to two dietary treatments consisting of a basal control diet (CON) or basal diet supplemented with 0.5% inulin (INU). Results showed that inulin supplementation tended to increase the average daily gain (ADG) at the fattening stage (0.05 < p < 0.10). Inulin significantly increased the dressing percentage (p < 0.05) and tended to increase the loin-eye area. The serum concentrations of insulin and IGF-I were significantly higher (p < 0.05) in the INU group than in the CON group. Moreover, inulin supplementation significantly elevated the expression level of myosin heavy chain II b (MyHC IIb) in the longissimus dorsi (p < 0.05). Inulin significantly upregulated the expression of mammalian rapamycin target protein (mTOR) but decreased (p < 0.05) the expression level of muscle-specific ubiquitin ligase MuRF-1. These results show the beneficial effect of inulin supplementation on the growth performance and carcass traits in growing–finishing pigs, and will also facilitate the application of inulin in swine production.

The protective effects of rapamycin on cell autophagy in the renal tissues of rats with diabetic nephropathy via mTOR-S6K1-LC3II signaling pathway

Background: Previous studies have shown that podocyte autophagy is an important trigger for proteinuria and glomerulosclerosis. The mammalian rapamycin target protein (mTOR) occupies a pivotal position in the autophagy pathway. In this study, we planned to clarify the mechanism of mTOR regulation of podocyte autophagy and the effect of rapamycin (RAPA).Methods: All rats were randomly divided into normal control group (n = 8), DN group (n = 8), and RAPA group (n = 8). Blood and urine samples were collected at the 4th, 8th, and 12th weeks of the experiment. The serum creatinine (Scr), urine volume levels, and the 24 h urine protein (UP) levels were examined. The nephrin, podocin, mTOR, ribosomal S6 kinase 1 (S6K1), and autophagy marker light chain 3 (LC3II) expression levels were evaluated by immunohistochemistry, quantitative PCR, and immunoblotting.Results: The urine volume, 24 h UP, and Scr of the DN and RAPA groups increased significantly compared with the NC group (p < .05). Nephrin and podocin expression was decreased in the kidney tissues of the DN and RAPA groups compared with the NC group (p < .05). The expression levels of mTOR and S6K1 increased and LC3II expression decreased in the renal tissues of the DN and RAPA groups compared with the NC group (p < .05). After RAPA treatment, all the above indexes were improved compared with the DN group (p < .05), but were significantly abnormal compared with the NC group (p < .05).Conclusion: The proteinuria and kidney function had improved after RAPA treatment. These results confirmed that RAPA specifically binds to mTOR kinase, and inhibits mTOR activity, thereby regulating the pathological autophagic process.

Advances in immunosuppressive mechanisms of rapamycin in aplastic anemia

Immunosuppressive therapy (IST) is one of the main treatment methods for aplastic anemia (AA). Rapamycin, as a mammalian rapamycin target protein (mTOR) inhibitor, exerts immunosuppression by inhibiting the activation of cytotoxic T lymphocyte(CTL), regulating the differentiation of T helper lymphocyte(Th), promoting the proliferation of regulatory T lymphocyte(Treg), and inhibiting the maturation and mobilization of dendritic cell(DC). Rapamycin provides new possibilities for the treatment of AA. In order to further explore the therapeutic strategies for AA, this article reviews literatures on immunosuppressive mechanisms of rapamycin in AA. Key words: Anemia, aplastic; Sirolimus; Immunosuppressive agents; Lymphocytes; Dendritic cells

Role of mTOR C1/C2 pathways involve in abrogation of chronic rejection in rat heart transplantation model

Objective To study the expression of the components of the mammalian rapamycin target protein (mTOR) pathway in T lymphocytes in rats with chronic rejection (CR), and to explore the role of mTOR pathway in the inhibition of CR. Methods ACI rat recipients received intraperitoneal ectopic cardiac transplantation with Wistar-Furth rat hearts. In the experimental group, a mutated class I major histocompatibility complex (MHC I) that can eliminate CR was delivered into recipients prior-operation, and a sub-therapeutic cyclosporine A (CsA) (10 mg/kg, 3 d) was also administered. In the experimental control group, the heart allograft recipients were treated with sub-therapeutic CsA (10 mg/kg, 3 d). The blank controls were the untreated recipients. Each group was divided into three subgroups (5 rats in each subgroup) according to the sacrifice time on the postoperative 1st, 3rd and 7th days. The spleen samples were taken for T cell extraction and Western blot analysis. Results Western blot results showed that rat heart allografts with abolished CR exhibited downregulation of the RAPA-sensitive mTORC1 elements including mTOR and Raptor, and down-regulation of the RAPA-insensitive mTORC2 elements including Rictor and Sin1. Conclusions Abrogation of CR in rat model system involves modulation of mTOR C1 and mTOR C2 pathways. The mTOR C1 pathway regulates cellular proliferation and the mTORC2 pathway regulates T-cell motility. Selective targeting of T-cell actin cytoskeletal pathways shows potential for pathway-targeted immunosuppression therapies. Key words: Chronic rejection; Transplantation; Immune tolerance; Actin cytoskeleton

Occurrence, diagnosis and treatment of de novo gastrointestinal malignancies after organ transplantation

With the continuous use of immunosuppressive agents routinely, the long-term survival rate of organ transplant recipients has been incessantly improved. However, the risk of de novo malignancies is also increasing, which has become the second cause of death after organ transplantation. De novo gastrointestinal malignancies are common after liver or kidney transplantation, mostly with advanced stage when diagnosed and poor prognosis. There is a significant trend in the development of de novo malignancies in transplant recipients, which is probably related to factors, including direct or indirect effects of immunosuppressive agents, precancerosis and survival time of transplanted grafts and recipients. Long-term postoperative use of immunosuppressive agents can keep the recipient's immune system in the inhibitory state, which provides the conditions for tumor cells to escape immune surveillance and to proliferate. In addition, some immunosuppressive agents [such as calcium phosphatase inhibitor (CNI)] are carcinogenic and the use of anti-tumor combined with immunosuppressive drugs can be considered [such as mycophenolate mofetil (MMF), mammalian rapamycin target protein (mTOR) inhibitors]. De novo gastrointestinal malignancy has no specific clinical manifestations. It is suggested that the recipients need to strictly comply with the follow-up time, avoid exposure to carcinogens, treat the precancerosis positively and have a medical examination carefully. At present, there are no literatures and guidelines about a standard treatment for de novo gastrointestinal malignancies after transplantation. The primary treatment of de novo malignancies should be adjusting the dosage of immunosuppressive agents to ensure that their minimum dose can effectively maintain the graft function. For patients with stable postoperative organ function and without acute or chronic rejection, the dosage of immunosuppressive agents can be reduced gradually to avoid an excessive immunosuppressive effect. Thereafter, the incidence of de novo malignancies is reduced to a minimum. Minimization of using CNI as soon as possible after transplantation has been widely recognized, and CNI plus mTOR inhibitor or MMF has become a relatively reasonable method.

Hippocampal endosomal, lysosomal, and autophagic dysregulation in mild cognitive impairment: correlation with aβ and tau pathology.

Endosomal-lysosomal and autophagic dysregulation occurs in the hippocampus in prodromal Alzheimer disease (AD), but its relationship with β-amyloid (Aβ) and tau pathology remains unclear. To investigate this issue, we performed immunoblot analysis of hippocampal homogenates from cases with an antemortem clinical diagnosis of no cognitive impairment, mild cognitive impairment (MCI), and AD. Western blot analysis revealed significant increases in the acid hydrolase cathepsin D and early endosome marker rabaptin5 in the MCI group compared with AD, whereas levels of phosphorylated mammalian target of rapamycin proteins (pmTOR), total mammalian target of rapamycin (mTOR), p62, traf6, and LilrB2 were comparable across clinical groups. Hippocampal Aβ1-40 and Aβ1-42 concentrations and AT8-immunopositive neurofibrillary tangle density were not significantly different across the clinical groups. Greater cathepsin D expression was associated with global cognitive score and episodic memory score but not with mini mental state examination or advanced neuropathology criteria. These results indicate that alterations in hippocampal endosomal-lysosomal proteins in MCI are independent of tau or Aβ pathology.

Raptor ablation in skeletal muscle decreases Cav1.1 expression and affects the function of the excitation-contraction coupling supramolecular complex.

The protein mammalian target of rapamycin (mTOR) is a serine/threonine kinase regulating a number of biochemical pathways controlling cell growth. mTOR exists in two complexes termed mTORC1 and mTORC2. Regulatory associated protein of mTOR (raptor) is associated with mTORC1 and is essential for its function. Ablation of raptor in skeletal muscle results in several phenotypic changes including decreased life expectancy, increased glycogen deposits and alterations of the twitch kinetics of slow fibres. In the present paper, we show that in muscle-specific raptor knockout (RamKO), the bulk of glycogen phosphorylase (GP) is mainly associated in its cAMP-non-stimulated form with sarcoplasmic reticulum (SR) membranes. In addition, 3[H]-ryanodine and 3[H]-PN200-110 equilibrium binding show a ryanodine to dihydropyridine receptors (DHPRs) ratio of 0.79 and 1.35 for wild-type (WT) and raptor KO skeletal muscle membranes respectively. Peak amplitude and time to peak of the global calcium transients evoked by supramaximal field stimulation were not different between WT and raptor KO. However, the increase in the voltage sensor-uncoupled RyRs leads to an increase of both frequency and mass of elementary calcium release events (ECRE) induced by hyper-osmotic shock in flexor digitorum brevis (FDB) fibres from raptor KO. The present study shows that the protein composition and function of the molecular machinery involved in skeletal muscle excitation-contraction (E-C) coupling is affected by mTORC1 signalling.