Humanβ-defensin-1 affects the mammalian target of rapamycin pathway and autophagy in colon cancer cells through long noncoding RNA TCONS_00014506

BACKGROUND Colorectal cancer has a low 5-year survival rate and high mortality.Humanβ-defensin-1(hBD-1)may play an integral function in the innate immune system,contributing to the recognition and destruction of cancer cells.Long non-coding RNAs(lncRNAs)are involved in the process of cell differentiation and growth.AIM To investigate the effect of hBD-1 on the mammalian target of rapamycin(mTOR)pathway and autophagy in human colon cancer SW620 cells.METHODS CCK8 assay was utilized for the detection of cell proliferation and determination of the optimal drug concentration.Colony formation assay was employed to assess the effect of hBD-1 on SW620 cell proliferation.Bioinformatics was used to screen potentially biologically significant lncRNAs related to the mTOR pathway.Additionally,p-mTOR(Ser2448),Beclin1,and LC3II/I expression levels in SW620 cells were assessed through Western blot analysis.RESULTS hBD-1 inhibited the proliferative ability of SW620 cells,as evidenced by the reduction in the colony formation capacity of SW620 cells upon exposure to hBD-1.hBD-1 decreased the expression of p-mTOR(Ser2448)protein and increased the expression of Beclin1 and LC3II/I protein.Furthermore,bioinformatics analysis identified seven lncRNAs(2 upregulated and 5 downregulated)related to the mTOR pathway.The lncRNA TCONS_00014506 was ultimately selected.Following the inhibition of the lncRNA TCONS_00014506,exposure to hBD-1 inhibited p-mTOR(Ser2448)and promoted Beclin1 and LC3II/I protein expression.CONCLUSION hBD-1 inhibits the mTOR pathway and promotes autophagy by upregulating the expression of the lncRNA TCONS_00014506 in SW620 cells.

MicroRNA-451 from Human Umbilical Cord-Derived Mesenchymal Stem Cell Exosomes Inhibits Alveolar Macrophage Autophagy via Tuberous Sclerosis Complex 1/Mammalian Target of Rapamycin Pathway to Attenuate Burn-Induced Acute Lung Injury in Rats

Objective Our previous studies established that microRNA(miR)-451 from human umbilical cord mesenchymal stem cell-derived exosomes(hUC-MSC-Exos)alleviates acute lung injury(ALI).This study aims to elucidate the mechanisms by which miR-451 in hUC-MSC-Exos reduces ALI by modulating macrophage autophagy.Methods Exosomes were isolated from hUC-MSCs.Severe burn-induced ALI rat models were treated with hUC-MSC-Exos carrying the miR-451 inhibitor.Hematoxylin-eosin staining evaluated inflammatory injury.Enzyme-linked immunosorbnent assay measured lipopolysaccharide(LPS),tumor necrosis factor-α,and interleukin-1βlevels.qRT-PCR detected miR-451 and tuberous sclerosis complex 1(TSC1)expressions.The regulatory role of miR-451 on TSC1 was determined using a dual-luciferase reporter system.Western blotting determined TSC1 and proteins related to the mammalian target of rapamycin(mTOR)pathway and autophagy.Immunofluorescence analysis was conducted to examine exosomes phagocytosis in alveolar macrophages and autophagy level.Results hUC-MSC-Exos with miR-451 inhibitor reduced burn-induced ALI and promoted macrophage autophagy.MiR-451 could be transferred from hUC-MSCs to alveolar macrophages via exosomes and directly targeted TSC1.Inhibiting miR-451 in hUC-MSC-Exos elevated TSC1 expression and inactivated the mTOR pathway in alveolar macrophages.Silencing TSC1 activated mTOR signaling and inhibited autophagy,while TSC1 knockdown reversed the autophagy from the miR-451 inhibitor-induced.Conclusion miR-451 from hUC-MSC exosomes improves ALI by suppressing alveolar macrophage autophagy through modulation of the TSC1/mTOR pathway,providing a potential therapeutic strategy for ALI.

Osteopontin promotes gastric cancer progression via phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway

BACKGROUND Gastric cancer(GC)is one of the most common malignant tumors.Osteopontin(OPN)is thought to be closely related to the occurrence,metastasis and prognosis of many types of tumors.AIM To investigate the effects of OPN on the proliferation,invasion and migration of GC cells and its possible mechanism.METHODS The mRNA and protein expression of OPN in the GC cells were analyzed by realtime quantitative-reverse transcription polymerase chain reaction and western blotting,and observe the effect of varying degree expression OPN on the proliferation and other behaviors of GC.Next,the effects of OPN knockdown on GC cells migration and invasion were examined.The short hairpin RNA(shRNA)and negative control shRNA targeting OPN-shRNA were transfected into the cells according to the manufacturer’s instructions.Non transfected cells were classified as control in the identical transfecting process.24 h after RNA transfection cell proliferation activity was detected by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide assay,and cell invasiveness and migration were detected by Trans well assay.Meanwhile,the expression of protein kinase B(AKT),matrix metalloproteinase 2(MMP-2)and vascular endothelial growth factor(VEGF)in the human GC cell lines was detected by reverse transcription polymerase chain reaction and western blotting.RESULTS The results of this study revealed that OPN mRNA and protein expression levels were highly expressed in SGC-7901 cells.OPN knockdown by specific shRNA noticeably reduced the capabilities of proliferation,invasion and migration of SGC-7901 cells.Moreover,in the experiments of investigating the underlying mechanism,results showed that OPN knockdown could down-regulated the expression of MMP-2 and VEGF,it also decreased the phosphorylation of AKT.Meanwhile,the protein expression levels of MMP-2,VEGF and phosphorylated AKT was noticeable lower than that in control group in the GC cells after they were added to phosphatidylinositol-3-kinase(PI3K)inhibitor(LY294002).CONCLUSION These results suggested that OPN though PI3K/AKT/mammalian target of rapamycin signal pathway to upregulate MMP-2 and VEGF expression,which contribute SGC-7901 cells to proliferation,invasion and migration.Thus,our results demonstrate that OPN may serve as a novel prognostic biomarkers as well as a potential therapeutic targets for GC.

Role of mammalian target of rapamycin complex 2 in primary and secondary liver cancer

The mammalian target of rapamycin(mTOR)acts in two structurally and functionally distinct protein complexes,mTOR complex 1(mTORC1)and mTOR complex 2(mTORC2).Upon deregulation,activated mTOR signaling is associated with multiple processes involved in tumor growth and metastasis.Compared with mTORC1,much less is known about mTORC2 in cancer,mainly because of the unavailability of a selective inhibitor.However,existing data suggest that mTORC2 with its two distinct subunits Rictor and mSin1 might play a more important role than assumed so far.It is one of the key effectors of the PI3K/AKT/mTOR pathway and stimulates cell growth,cell survival,metabolism,and cytoskeletal organization.It is not only implicated in tumor progression,metastasis,and the tumor microenvironment but also in resistance to therapy.Rictor,the central subunit of mTORC2,was found to be upregulated in different kinds of cancers and is associated with advanced tumor stages and a bad prognosis.Moreover,AKT,the main downstream regulator of mTORC2/Rictor,is one of the most highly activated proteins in cancer.Primary and secondary liver cancer are major problems for current cancer therapy due to the lack of specific medical treatment,emphasizing the need for further therapeutic options.This review,therefore,summarizes the role of mTORC2/Rictor in cancer,with special focus on primary liver cancer but also on liver metastases.

Mammalian target of rapamycin;novel insight for management of inflammatory bowel diseases

Inflammatory bowel diseases(IBDs),with blurred etiology,show a rising trend and are of global concern.Of various factors involved in IBD pathogenesis and development,inflammation has been shown to play a major role.Recognition of the molecular and cellular pathways that induce IBD is an emerging subject to develop targeted therapies.Mammalian target of rapamycin(mTOR)is one the most common receptors of many inflammatory pathways,including that of IBD.To this end,we intend to overview the mTOR inhibitors for their possible efficacy in present and future approaches to treatment of IBD.

Production of interleukin-1β related to mammalian target of rapamycin/Toll-like receptor 4 signaling pathway during Aspergillus fumigatus infection of the mouse cornea

AIM：To elucidate the effect of rapamycin on regulating the production of interleukin（IL）-1β in Aspergillus fumigatus（A.fumigatus）-induced keratitis and to verify whether the expression of IL-1β in A.fumigatus keratitis is associated with the mammalian target of rapamycin（mT OR）/Toll-like receptor 4（TLR4） signaling pathway.METHODS：Fungal keratitis mouse models of susceptible C57 BL/6 mice were established using A.fumigatus.The mice were subsequently treated with rapamycin.The protein levels of p-mT OR,TLR4,and IL-1β in normal and infected corneal tissue were measured by Western blot.The TLR4 and IL-1β m RNA levels were determined by real-time polymerase chain reaction（PCR）.RESULTS：In C57 BL/6 mice,rapamycin treatment decreased the clinical scores and production of the pro-inflammatory cytokine,IL-1β.The expression of TLR4,stimulated by A.fumigatus,was reduced as well when the mT OR signaling pathway was suppressed by rapamycin.CONCLUSION：Rapamycin is beneficial for the outcome of fungal keratitis and has an inhibitory effect expression of the inflammatory cytokine IL-1β.The inhibitory effect on IL-1β expression can be associated with the mT OR/TLR4 signaling pathway in A.fumigatus infection in mice.

Cytotoxicity of nonylphenol on spermatogonial stem cells via phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin pathway

BACKGROUND With continuous advancement of industrial society,environmental pollution has become more and more serious.There has been an increase in infertility caused by environmental factors.Nonylphenol(NP)is a stable degradation product widely used in daily life and production and has been proven to affect male fertility.However,the underlying mechanisms therein are unclear.Thus,it is necessary to study the effect and mechanism of NP on spermatogonial stem cells(SSCs).AIM To investigate the cytotoxic effect of NP on SSCs via the phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/AKT/mTOR)pathway.METHODS SSCs were treated with NP at 0,10,20 or 30μmol.MTT assay was performed to evaluate the effect of NP on the proliferation of SSCs.Flow cytometry was conducted to measure SSC apoptosis.The expression of Bad,Bcl-2,cytochrome-c,pro-Caspase 9,SOX-2,OCT-4,Nanog,Nanos3,Stra8,Scp3,GFRα1,CD90,VASA,Nanos2,KIT,PLZF and PI3K/AKT/mTOR-related proteins was observed by western blot,and the mRNA expression of SOX-2,OCT-4 and Nanog was detected by quantitative reverse transcription polymerase chain reaction.RESULTS Compared with untreated cells(0μmol NP),SSCs treated with NP at all concentrations showed a decrease in cell proliferation and expression of Bcl-2,Nanog,OCT-4,SOX-2,Nanos3,Stra8,Scp3,GFRα1,CD90,VASA,Nanos2,KIT,and PLZF(P<0.05),whereas the expression of Bad,cytochrome-c,and pro-Caspase 9 increased significantly(P<0.05).We further examined the PI3K/AKT/mTOR pathway and found that the phosphorylation of PI3K,AKT,mTORC1,and S6K was significantly decreased by NP at all concentrations compared to that in untreated SSCs(P<0.05).NP exerted the greatest effect at 30μmol among all NP concentrations.CONCLUSION NP attenuated the proliferation,differentiation and stemness maintenance of SSCs while promoting apoptosis and oxidative stress.The associated mechanism may be related to the PI3K/AKT/mTOR pathway.

Mammalian target of rapamycin complex 1 as an inducer of neurotrophic factors in dopaminergic neurons

The defining neuropathological feature of Parkinson＇s disease （PD） is the loss of nigrostriatal dopaminergic （DA） projections. This results in striatal dopamine levels and a biochemical reduction of movement disorders, such as a tremor at rest, rigidity of the limbs, bradykinesia, and postural instability （Kim et al., 2011; Kim et al., 2012; Burke and O＇Malley, 2013; Leem et al., 2014; Namet al., 2014）.

The Expression of Mammalian Target of Rapamycin in Ishikawa and HEC-1A Cells

The activation of mammalian target of rapamycin （mTOR） signaling pathway in endometrial carcinoma cells Ishikawa and HEC-1A was investigated. The expression of mTOR was detected by confocal fluorescence microscopy in Ishikawa and HEC-1A cells. The mRNA levels of PTEN and mTOR, the downstream substrate S6K1 and 4E-BP1 protein were assayed by RT-PCR and Western blot, respectively. The expression of PTEN in Ishikawa cells was deficient, but intact in HEC-1A cells respectively （P〈0.01）. There was mTOR expression in both Ishikawa and HEC-1A cells and the phosporylated substrate levels in Ishikawa cells were higher than those in HEC-1A cells （P〈0.05）. mTOR signaling pathway is activated in two endometrial carcinoma cell strains and the status of activation is related with PTEN expression of the cells. The activation level of mTOR is higher in PTEN-deficient endometrial carcinoma cells than that in PTEN-intact endometrial carcinoma cells.

Inactivation of mammalian target of rapamycin （mTOR） by rapamycin in a murine model of lipopolysaccharide-induced acute lung injury

Background The mammalian target of rapamycin （mTOR） pathway, a key cellular signaling pathway associated with various cellular functions, has distinct roles in the inflammatory process. In this study, the mTOR inhibitor rapamycin （Rapa） was used to test whether inhibition of mTOR activation attenuates lipopolysaccharide （LPS）-induced acute lung injury （ALl） in a murine model.Methods Mice pretreated with Rapa or vehicle were given LPS intratracheally. Local cell numbers and inflammatory cytokines present in the bronchoalveolar lavage fluid （BAL）, wet-to-dry weight ratio, histopathology of the lungs, and survival were evaluated.Results The phosphorylation of S6, a major downstream target of mTOR, had a 3-fold increase in lung tissue after LPS stimulation, but the increase was blocked by Rapa. Rapa reduced the levels of TNF-α （LPS vs. LPS ＋ Rapa,（1672.74±193.73） vs. （539.17±140.48） pg/ml, respectively; P 〈0.01） and IL-6 （LPS vs. LPS ＋ Rapa： （7790.88±1170.54）vs. （1968.57±474.62） pg/ml, respectively; P 〈0.01） in the BAL fluid. However, Rapa had limited effects on the overall severity of ALI, as determined by the wet-to-dry weight ratio of the lungs, number of neutrophils in the BAL fluid, and changes in histopathology. In addition, Rapa failed to reduce mortality in the LPS-induced ALI model.Conclusions We confirmed that mTOR was activated during LPS-induced ALI and strongly inhibited by Rapa.Although Rapa reduced the levels of the mediators of inflammation, the overall severity and survival of the ALI murine model were unchanged.

Chronic intermittent hypoxia increases β cell mass and activates the mammalian target of rapamycin/hypoxia inducible factor 1/vascular endothelial growth factor A pathway in mice pancreatic islet

Background Growing evidence from population and clinic based studies showed that obstructive sleep apnea （OSA） and its characterizing chronic intermittent hypoxia （IH） were independently associated with the development of type 2 diabetes mellitus.However,the pathogenesis by which OSA induces glucose metabolic disorders is not clear.We determined changes in pancreatic β cell mass and the mammalian target of rapamycin （mTOR）/hypoxia inducible factor 1 （HIF-1）/ vascular endothelial growth factor A （VEGF-A） pathway following IH exposure.Methods A controlled gas delivery system regulated the flow of nitrogen and oxygen into a customized cage housing mice during the experiment.Twenty-four male wild C57BL/6J mice were either exposed to IH （n=12） or intermittent air as a control （n=12） for 56 days.Mice were anaesthetized and sacrificed after exposure,pancreas samples were dissected for immunofluorescent staining.Insulin and DAPI staining labelled islet β cells.Insulin positive area and β cell number per islet were measured.P-S6,HIF-1α and VEGF-A staining were performed to detect the activation of mTOR/HIF-1NEGF-A pathway.Results After eight weeks of IH exposure,insulin positive area increased by an average of 18.5% （P 〈0.05）.The β cell number per islet increased （92 vs.55,respectively for IH and the control groups,P 〈0.05） with no change in the size of individual β cells.Islet expression of HIF-1α and VEGF-A were higher in IH group than control group,and percentage of p-S6 positive β cell also increased after IH exposure （16.8% vs.4.6% respectively for IH and the control groups,P 〈0.05）.Conclusion The number of pancreatic β cells increased as did the activity of the mTOR/HIF-1NEGF-A pathway after exposure to IH.

Accelerated Autophagy of Cecal Ligation and Puncture-Induced Myocardial Dysfunction and Its Correlation with Mammalian Target of Rapamycin Pathway in Rats

Background：Recent studies have indicated that autophagy is involved in sepsis-induced myocardial dysfunction.This study aimed to investigate the change of autophagy in cecal ligation and puncture （CLP）-induced myocardium dysfunction and its relationship with mammalian target of rapamycin （mTOR） pathway.Methods：Totally,12 rats were randomly divided into CLP group or sham-operated （SHAM） group.Cardiac tissues were harvested 18 h after CLP or sham operation.Pathology was detected by hematoxylin and eosin staining,cardiac functions by echocardiography,distribution ofmicrotubule-associated protein light chain 3 type Ⅱ （LC3II） by immunohistochemical staining,and autophagic vacuoles by transmission electron microscopy.Moreover,phosphorylation of mTOR （p-mTOR）,phosphorylation of S6 kinase-1 （PS6K1）,and LC3II and p62 expression were measured by western blotting.Pearson＇s correlation coefficient was used to analyze the correlation of two parameters.Results：The results by pathology and echocardiography revealed that there was obvious myocardial injury in CLP rats （left ventricle ejection fraction：SHAM 0.76 ± 0.06 vs.CLP 0.59 ± 0.l l,P 〈 0.01;fractional shortening：SHAM 0.51 ± 0.09 vs.CLP 0.37 ± 0.06,P 〈 0.05）.We also found that the autophagy process was elevated by CLE the ratio of LC3II/LC3I was increased （P 〈 0.05） while the expression of p62 was decreased （P 〈 0.05） in the CLP rats,and there were also more autophagosomes and autolysosomes in the CLP rats.Furthermore,the mTOR pathway in CLP myocardium was inhibited when compared with the sham-operated rats;p-mTOR （P 〈 0.01） and PS6K 1 （P 〈 0.05） were both significantly suppressed following CLP challenge.Interestingly,we found that the mTOR pathway was closely correlated with the autophagy processes.In our study,while p-mTOR in the myocardium was significantly correlated with p62 （r=0.66,P =0.02）,PS6K1 was significantly positively correlated with p62 （r =0.70,P =0.01） and negatively correlated with LC31I （r =-0.71,P =0.01）.Conclusions：The autophagy process in the myocardium was accelerated in CLP rats,which was closely correlated with the inhibition of the mTOR pathway.

Influence of Phosphatidylinositol-3-Kinase/Protein Kinase B-Mammalian Target of Rapamycin Signaling Pathway on the Neuropathic Pain Complicated by Nucleoside Reverse Transcriptase Inhibitors for the Treatment of HIV Infection

Background： Nucleoside reverse transcriptase inhibitors （NRTIs） are the earliest and most commonly used anti-human immunodeficiency virus drugs and play an important role in high active antiretroviral therapy. However, NRTI drug therapy can cause peripheral neuropathic pain. In this study, we aimed to investigate the mechanisms ofrapamycin on the pain sensitization of model mice by in vivo experiments to explore the effect of mammalian target of rapamycin （mTOR） in the pathogenesis ofneuropathic pain caused by NRTIs. Methods： Male Kun Ming （KM） mice weighing 20-2 g were divided into control, 2 mg/kg rapamycin, 12 mg/kg stavudine, and CMC-Na groups. Drugs were orally administered to mice for 42 consecutive days. The von Frey filament detection and thermal pain tests were conducted on day 7, 14, 21, 28, 35, and 42 after drug administration. After the last behavioral tests, immunohistochemistry and western blotting assay were used for the measurement of mTOR and other biomarkers. Multivariate analysis of variance was used. Results： The beneficial effects ofrapamycin on neuropathic pain were attributed to a reduction in mammalian target of rapamycin sensitive complex 1 （mTORC1）-positive cells （70.80± 2.41 vs. 112.30 ± 5.66, F = 34.36, P 〈 0.01 ） and mTORC1 activity in the mouse spinal cord. Mechanistic studies revealed that Protein Kinase B （Akt）/mTOR signaling pathway blockade with rapamycin prevented the phosphorylation of mTORC1 in stavudine-intoxicated mice （0.72 ± 0.04 vs. 0.86 ± 0.03, F=4.24, P = 0.045）, as well as decreased the expression of phospho-pTOS6K （0.47 ± 0.01 vs. 0.68 ± 0.03, F=6.01, P = 0.022） and phospho-4EBP1 （0.90 ± 0.04 vs. 0.94 ± 0.06, F= 0.28, P = 0.646）. Conclusions： Taken together, these results suggest that stavudine elevates the expression and activity of mTORC1 in the spinal cord through activating the Akt/mTOR signaling pathway. The data also provide evidence that rapamycin might be useful for the treatment of peripheral neuropathic pain.

TopoisomeraseⅡalpha promotes gallbladder cancer proliferation and metastasis through activating phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway

Background:TopoisomeraseⅡalpha(TOP2A)has been reported to play a crucial role in the tumorigenesis of various cancer types.However,the biological role of TOP2A in gallbladder cancer(GBC)remains unknown.The current study aimed to explore the function and potential mechanism of TOP2A in GBC.Methods:Based on Gene Expression Profiling Interactive Analysis data,we found TOP2A was significantly up-regulated in GBC tissues and resulting in shorter overall survival.Quantitative real-time polymerase chain reaction and immunohistochemistry were conducted to detect the expression of TOP2A in 45 pairs of GBC tissues and adjacent non-tumor tissues.In vitro,cell proliferation,migration,and invasion ability were examined by cell counting kit-8 and transwell assay,respectively.Epithelial-mesenchymal transition(EMT)related and phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)pathway-related markers were measured by Western blotting.Xenograft model assay was performed to evaluate the effect of TOP2A in vivo.Results:TOP2A was found up-regulated in GBC(tumor vs.normal,12.62 vs.0.34)and correlated with the late tumor node metastasis stage(P=0.0032),present of lymph node metastasis(P=0.0273),and poor prognosis in GBC patients(log-rank P=0.028).In vitro and in vivo assays showed that knockdown of TOP2A notably inhibited cell proliferation,migration,invasion,EMT process,and tumor growth in GBC.In addition,TOP2A down-regulation significantly decreased the protein levels of phosphor(p)-PI3K,p-Akt,and p-mTOR.Conclusion:Our study demonstrates that TOP2A was overexpressed in GBC and associated with poor prognosis in GBC patients.TOP2A promotes GBC cell proliferation,migration,invasion,EMT process,and tumor growth through activating PI3K/Akt/mTOR signaling pathway,and may serve as a novel prognostic biomarker and therapeutic target for GBC.

Targeting the mammalian target of rapamycin pathway in osteosarcoma using combinative chemotherapy

Osteosarcoma is the most common primary malignant ＇bone sarcoma, and occurs predominantly in childrenand young adults. The overall survival of patients with osteosarcoma remains low at approximately 60%.1 The prognosis of osteosarcoma patients is highly associated with response to chemotherapy. Although multiple chemotherapy regimens have improved the outcome of patients with osteosarcoma, resistance to current regimens has been reported in more than 30% of patients,2 highlighting the need for novel, targeted therapies.

A selectively suppressing amino acid transporter: Sodium-coupled neutral amino acid transporter 2 inhibits cell growth and mammalian target of rapamycin complex 1 pathway in skeletal muscle cells

Sodium-coupled neutral amino acid transporter 2(SNAT2),also known as solute carrier family 38 member 2(SLC38 A2),is expressed in the skeletal muscle.Our research previously indicated that SNAT2 m RNA expression level in the skeletal muscle was modulated by genotype and dietary protein.The aim of this study was to investigate the key role of the amino acid transporter SNAT2 in muscle cell growth,differentiation,and related signaling pathways via SNAT2 suppression using the inhibitor a-methylaminoisobutyric acid(Me AIB).The results showed that SNAT2 suppression down-regulated both the m RNA and protein expression levels of SNAT2 in C2 C12 cells,inhibited cell viability and differentiation of the cell,and regulated the cell distribution in G0/G1 and S phases(P<0.05).Meanwhile,most of the intercellular amino acid content of the cells after Me AIB co-culturing was significantly lower(P<0.05).Furthermore,the m RNA expression levels of system L amino acid transporter 1(LAT1),silent information regulator 1,and peroxisome proliferator-activated receptor-gamma co-activator 1 alpha,as well as the protein expression levels of amino acid transporters LAT1 and vacuolar protein sorting 34,were all down-regulated.The phosphorylated protein expression levels of mammalian target of rapamycin(m TOR),regulatory-associated protein of m TOR,4 E binding protein 1,and ribosomal protein S6 kinase 1 after Me AIB treatment were also significantly down-regulated(P<0.05),which could contribute to the importance of SNAT2 in amino acid transportation and skeletal muscle cell sensing.In conclusion,SNAT2 suppression inhibited C2 C12 cell growth and differentiation,as well as the availability of free amino acids.Although the m TOR complex 1 signaling pathway was found to be involved,its response to different nutrients requires further study.

Statin, aspirin and metformin use and risk of hepatocellular carcinoma related outcomes following liver transplantation: A retrospective study

BACKGROUND Liver transplantation(LT)is a potentially curative therapy for patients with hepatocellular carcinoma(HCC).HCC-recurrence following LT is associated with reduced survival.There is increasing interest in chemoprophylaxis to improve HCC-related outcomes post-LT.AIM To investigate whether there is any benefit for the use of drugs with proposed chemoprophylactic properties against HCC,and patient outcomes following LT.METHODS This was a retrospective study of adult patients who received Deceased Donor LT for HCC from 2005-2022,from a single Australian centre.Drug use was defined as statin,aspirin or metformin therapy for≥29 days,within 24 months post-LT.A cox proportional-hazards model with time-dependent covariates was used for survival analysis.Outcome measures were the composite-endpoint of HCC-recurrence and all-cause mortality,HCC-recurrence and HCC-related mortality.Sensitivity analysis was performed to account for immortality time bias and statin dosing.RESULTS Three hundred and five patients were included in this study,with 253(82.95%)males with a median age of 58.90 years.Aetiologies of liver disease were 150(49.18%)hepatitis C,73(23.93%)hepatitis B(HBV)and 33(10.82%)non-alcoholic fatty liver disease(NAFLD).56(18.36%)took statins,51(16.72%)aspirin and 50(16.39%)metformin.During a median follow-up time of 59.90 months,34(11.15%)developed HCC-recurrence,48(15.74%)died,17(5.57%)from HCC-related mortality.Statin,aspirin or metformin use was not associated with statistically significant differences in the composite endpoint of HCC-recurrence or all-cause mortality[hazard ratio(HR):1.16,95%CI:0.58-2.30;HR:1.21,95%CI:0.28-5.27;HR:0.61,95%CI:0.27-1.36],HCC-recurrence(HR:0.52,95%CI:0.20-1.35;HR:0.51,95%CI:0.14-1.93;HR 1.00,95%CI:0.37-2.72),or HCC-related mortality(HR:0.32,95%CI:0.033-3.09;HR:0.71,95%CI:0.14-3.73;HR:1.57,95%CI:0.61-4.04)respectively.Statin dosing was not associated with statist-ically significant differences in HCC-related outcomes.CONCLUSION Statin,metformin or aspirin use was not associated with improved HCC-related outcomes post-LT,in a largely historical cohort of Australian patients with a low proportion of NAFLD.Further prospective,multicentre studies are required to clarify any potential benefit of these drugs to improve HCC-related outcomes.

Inhibitory Effect of PPARδAgonist GW501516 on Proliferation of Hypoxia-induced Pulmonary Arterial Smooth Muscle Cells by Regulating the mTOR Pathway

Objective This study aimed to investigate the effects of the peroxisome proliferator-activated receptorδ(PPARδ)agonist GW501516 on the proliferation of pulmonary artery smooth muscle cells(PASMCs)induced by hypoxia,in order to search for new drugs for the treatment and prevention of pulmonary vascular remodeling.Methods PASMCs were incubated with different concentrations of GW501516(10,30,100 nmol/L)under the hypoxic condition.The proliferation was determined by a CCK-8 assay.The cell cycle progression was analyzed by flow cytometry.The expression of PPARδ,S phase kinase-associated protein 2(Skp2),and cell cycle-dependent kinase inhibitor p27 was detected by Western blotting.Then PASMCs were treated with 100 nmol/L GW501516,100 nmol/L mammalian target of rapamycin(mTOR)inhibitor rapamycin and/or 2µmol/L mTOR activator MHY1485 to explore the molecular mechanisms by which GW501516 reduces the proliferation of PASMCs.Results The presented data demonstrated that hypoxia reduced the expression of PPARδin an oxygen concentration-and time-dependent manner,and GW501516 decreased the proliferation of PASMCs induced by hypoxia by blocking the progression through the G0/G1 to S phase of the cell cycle.In accordance with these findings,GW501516 downregulated Skp2 and upregulated p27 in hypoxia-exposed PASMCs.Further experiments showed that rapamycin had similar effects as GW501516 in inhibiting cell proliferation,arresting the cell cycle,regulating the expression of Skp2 and p27,and inactivating mTOR in hypoxia-exposed PASMCs.Moreover,MHY1485 reversed all the beneficial effects of GW501516 on hypoxia-stimulated PASMCs.Conclusion GW501516 inhibited the proliferation of PASMCs induced by hypoxia through blocking the mTOR/Skp2/p27 signaling pathway.

Intracellular accumulation of tau inhibits autophagosome formation by activating TIA1-amino acid-mTORC1 signaling

Background:Autophagy dysfunction plays a crucial role in tau accumulation and neurodegeneration in Alzheimer’s disease(AD).This study aimed to investigate whether and how the accumulating tau may in turn affect autophagy.Methods:The primary hippocampal neurons,N2a and HEK293T cells with tau overexpression were respectively starved and treated with vinblastine to study the effects of tau on the initiating steps of autophagy,which was analysed by Student’s two-tailed t-test.The rapamycin and concanamycin A were employed to inhibit the mammalian target of rapamycin kinase complex 1(mTORC1)activity and the vacuolar H+-ATPase(v-ATPase)activity,respectively,which were analysed by One-way ANOVA with post hoc tests.The Western blotting,co-immunoprecipitation and immunofuorescence staining were conducted to gain insight into the mechanisms underlying the tau effects of mTORC1 signaling alterations,as analysed by Student’s two-tailed t-test or One-way ANOVA with post hoc tests.The autophagosome formation was detected by immunofuorescence staining and transmission electron microscopy.The amino acids(AA)levels were detected by high performance liquid chromatography(HPLC).Results:We observed that overexpressing human full-length wild-type tau to mimic AD-like tau accumulation induced autophagy deficits.Further studies revealed that the increased tau could bind to the prion-related domain of T cell intracellular antigen 1(PRD-TIA1)and this association significantly increased the intercellular level of amino acids(Leucine,P=0.0038;Glutamic acid,P=0.0348;Alanine,P=0.0037;Glycine,P=0.0104),with concordant upregulation of mTORC1 activity[phosphorylated eukaryotic translation initiation factor 4E-binding protein 1(p-4EBP1),P<0.0001;phosphorylated 70 kD ribosomal protein S6 kinase 1(p-p70S6K1),P=0.0001,phosphorylated unc-51-like autophagyactivating kinase 1(p-ULK1),P=0.0015]and inhibition of autophagosome formation[microtubuleassociated protein light chain 3 II(LC3 II),P=0.0073;LC3 puncta,P<0.0001].As expected,this tau-induced deficit of autophagosome formation in turn aggravated tau accumulation.Importantly,we also found that blocking TIA1 and tau interaction by overexpressing PRD-TIA1,downregulating the endogenous TIA1 expression by shRNA,or downregulating tau protein level by a small proteolysis targeting chimera(PROTAC)could remarkably attenuate tau-induced autophagy impairment.Conclusions:Our findings reveal that AD-like tau accumulation inhibits autophagosome formation and induces autophagy deficits by activating the TIA1/amino acid/mTORC1 pathway,and thus this work reveals new insight into tau-associated neurodegeneration and provides evidence supporting the use of new therapeutic targets for AD treat-ment and that of related tauopathies.

Dexmedetomidine mediates the mechanism of action of ferroptosis in mice with Alzheimer’s disease by regulating the mTOR-TFR1 pathway

BACKGROUND Alzheimer’s disease(AD)is the most common neurodegenerative disorder,and there are currently no effective drugs to delay progression of the disease.Ferroptosis may play a vital part in AD,and is therefore receiving increasing attention by researchers.AIM To investigate the effects of dexmedetomidine(Dex)on ferroptosis in AD mouse hippocampus.METHODS Hippocampal neurons(HNs)HT22 were induced by amyloidβ-protein(Aβ)and both in vitro and in vivo AD mouse models were prepared via injections.The cellcounting kit-8 assay and immunofluorescence technique were adopted to determine cell proliferation activity and intracellular Fe2+levels,and the TBA method and microplate method were employed for malondialdehyde and glutathione measurements,respectively.Hippocampal tissue damage was determined using hematoxylin and eosin and Nissl staining.Mouse learning and memory ability in each group was assessed by the Morris water maze test,and the expression levels of mammalian target of rapamycin(mTOR)signal molecules and ferroptosis-related proteins transferrin receptor 1(TFR1),SLC7A11 and glutathione peroxidase 4 were examined by western blotting.RESULTS Dex enhanced lipid peroxidation and iron influx in mouse HNs in both in vitro and in vivo experiments,while inhibition of the mTOR axis blocked this process.These findings demonstrate that Dex can inhibit ferroptosis-induced damage in mouse HNs by activating mTOR-TFR1 signaling to regulate ferroptosis-associated proteins,thus alleviating cognitive dysfunction in AD mice.CONCLUSION Dex can activate the mTOR-TFR1 axis to inhibit ferroptosis in mouse HNs,thereby improving the learning and memory ability of mice.