mTOR Protein Levels Research Articles

Tumor suppressive miR-99b-5p as an epigenomic regulator mediating mTOR/AR/SMARCD1 signaling axis in aggressive prostate cancer.

African American (AA) men exhibited 2.3-fold higher PCa incidence and 1.7-fold higher PCa mortality rates when compared to the European American (EA) men. Besides the socioeconomic factors, emerging evidence has highlighted that biological risk factors may play critical roles in the AA PCa disparities. Previously, we have shown that downregulated miR-99b-5p and upregulated mTOR cooperatively promotes the AA PCa aggressiveness and drug resistance. In this study, we aimed to explore the miR-99b-5p/mTOR/AR/SMARCD1 signaling axis in AA PCa aggressiveness. The analyses used in the study included immunofluorescence, western blot, in-vitro functional assays (TUNEL, colony forming, and MTT), and chromatin immunoprecipitation (ChIP)-qPCR assays in 2D and/or 3D culture model of EA PCa and AA PCa cell lines. Specifically, the immunofluorescence staining, and western blot analysis has revealed that nuclear mTOR, AR, and SMARCD1 were highly expressed in AA PCa (MDA PCa 2b) compared to EA PCa (LNCaP) cell line. Western blot analysis further revealed that miR-99b-5p inhibited protein levels of mTOR, AR/AR-V7 and SMARCD1 in cytoplasm and nuclei of EA and AA PCa. The in-vitro functional (MTT, TUNEL, and clonogenic) assays have demonstrated that miR-99b-5p effectively inhibited cell proliferation/survival and induced cell apoptosis in EA and AA PCa cells. Moreover, combination of miR-99b-5p and enzalutamide (Enz) synergistically enhances the cytotoxicity against aggressive AA PCa and castration-resistant prostate cancer (CRPC). mTOR ChIP-qPCR assays further demonstrated that miR-99b-5p or miR-99b-5p/Enz significantly reduces the recruitment of mTOR to the genes involved in the metabolic reprogramming in CRPC. Taken together, miR-99b-5p may function as an epigenomic driver to modulate the mTOR/AR/SMARCD1 signaling axis in AA PCa and resistant CRPC.

METTL3 regulates glucose transporter expression in placenta exposed to hyperglycemia through the mTOR signaling pathway.

Alterations in the placental expression of glucose transporters (GLUTs), the crucial maternal-fetal nutrient transporters, have been found in women with hyperglycemia in pregnancy (HIP). However, there is still uncertainty about the underlying effect of the high-glucose environment on placental GLUTs expression in HIP. We quantitatively evaluated the activity of mammalian target of rapamycin (mTOR) and expression of GLUTs (GLUT1, GLUT3, and GLUT4) in the placenta of women with normal pregnancies (CTRL, n = 12) and pregnant women complicated with poorly controlled type 2 diabetes mellitus (T2DM, n = 12) by immunohistochemistry. In addition, BeWo cells were treated with different glucose concentrations to verify the regulation of hyperglycemia. Then, changes in the expression of GLUTs following the activation or suppression of the mTOR pathway were also assessed using MHY1485/rapamycin (RAPA) treatment or small interfering RNA (siRNA)-mediated silencing approaches. Moreover, we further explored the alteration and potential upstream regulatory role of methyltransferase-like 3 (METTL3) when exposed to hyperglycemia. mTOR, phosphorylated mTOR (p-mTOR), and GLUT1 protein levels were upregulated in the placenta of women with T2DM compared with those CTRL. In BeWo cells, mTOR activity increased with increasing glucose concentration, and the expression of GLUT1, GLUT3, and GLUT4 as well as GLUT1 cell membrane translocation were upregulated by hyperglycemia to varying degrees. Both the drug-mediated and genetic depletion of mTOR signaling in BeWo cells suppressed GLUTs expression, whereas MHY1485-induced mTOR activation upregulated GLUTs expression. Additionally, high glucose levels upregulated METTL3 expression and nuclear translocation, and decreasing METTL3 levels suppressed GLUTs expression and mTOR activity and vice versa . Furthermore, in METTL3 knockdown BeWo cells, the inhibitory effect on GLUTs expression was eliminated by activating the mTOR signaling pathway using MHY1485. High-glucose environment-induced upregulation of METTL3 in trophoblasts regulates the expression of GLUTs through mTOR signaling, contributing to disordered nutrient transport in women with HIP.

Diosgenin alleviates NAFLD induced by a high-fat diet in rats via mTOR/SREBP-1c/HSP60/MCAD/SCAD signaling pathway

This study aims to observe the effects of diosgenin on the expression of mammalian target of rapamycin(mTOR), sterol regulatory element-binding protein-1c(SREBP-1c), heat shock protein 60(HSP60), medium-chain acyl-CoA dehydrogenase(MCAD), and short-chain acyl-CoA dehydrogenase(SCAD) in the liver tissue of the rat model of non-alcoholic fatty liver disease(NAFLD) and explore the mechanism of diosgenin in alleviating NAFLD. Forty male SD rats were randomized into five groups: a control group, a model group, low-(150 mg·kg~(-1)·d~(-1)) and high-dose(300 mg·kg~(-1)·d~(-1)) diosgenin groups, and a simvastatin(4 mg·kg~(-1)·d~(-1)) group. The rats in the control group were fed with a normal diet, while those in the other four groups were fed with a high-fat diet. After feeding for 8 weeks, the body weight of rats in the high-fat diet groups increased significantly. After that, the rats were administrated with the corresponding dose of diosgenin or simvastatin by gavage every day for 8 weeks. The levels of triglyceride(TG), total cholesterol(TC), alanine transaminase(ALT), and aspartate transaminase(AST) in the serum were determined by the biochemical method. The levels of TG and TC in the liver were measured by the enzyme method. Oil-red O staining was employed to detect the lipid accumulation, and hematoxylin-eosin(HE) staining to detect the pathological changes in the liver tissue. The mRNA and protein levels of mTOR, SREBP-1c, HSP60, MCAD, and SCAD in the liver tissue of rats were determined by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR) and Western blot, respectively. Compared with the control group, the model group showed increased body weight, food uptake, liver index, TG, TC, ALT, and AST levels in the serum, TG and TC levels in the liver, lipid deposition in the liver, obvious hepatic steatosis, up-regulated mRNA and protein expression levels of mTOR and SREBP-1c, and down-regulated mRNA and protein expression levels of HSP60, MCAD, and SCAD. Compared with the model group, the rats in each treatment group showed obviously decreased body weight, food uptake, liver index, TG, TC, ALT, and AST levels in the serum, TG and TC levels in the liver, lessened lipid deposition in the liver, ameliorated hepatic steatosis, down-regulated mRNA and protein le-vels of mTOR and SREBP-1c, and up-regulated mRNA and protein levels of HSP60, MCAD, and SCAD. The high-dose diosgenin outperformed the low-dose diosgenin and simvastatin. Diosgenin may prevent and treat NAFLD by inhibiting the expression of mTOR and SREBP-1c and promoting the expression of HSP60, MCAD, and SCAD to reduce lipid synthesis, improving mitochondrial function, and promoting fatty acid β oxidation in the liver.

Effects of Vitamin D3 Supplementation and Aerobic Training on Autophagy Signaling Proteins in a Rat Model Type 2 Diabetes Induced by High-Fat Diet and Streptozotocin.

The aim of this study was to investigate the combined effects of vitamin D3 supplementation and aerobic training on regulating the autophagy process in rats with type 2 diabetic induced by a high-fat diet and streptozotocin. A total of 40 Wistar rats were divided into five groups: normal control (NC), diabetic control (DC), diabetic + aerobic training (DAT), diabetic + vitamin D3 (DVD), and diabetic + aerobic training + vitamin D3 (DVDAT). The rats underwent eight weeks of aerobic training with an intensity of 60% maximum running speed for one hour, along with weekly subcutaneous injections of 10,000 units of vitamin D3. The protein levels of different autophagy markers were assessed in the left ventricular heart tissue. The results showed that the protein levels of AMPK, pAMPK, mTOR, and pmTOR were significantly lower in the DC group compared to the NC group. Conversely, the levels of ULK, Beclin-1, LC3II, Fyco, and Cathepsin D proteins were significantly higher in the DC group. However, the interventions of aerobic training and vitamin D3 supplementation, either individually or in combination, led to increased levels of AMPK, pAMPK, mTOR, and pmTOR, and decreased levels of ULK, Beclin-1, LC3II, Fyco, and Cathepsin D (p < 0.05). Additionally, the aerobic capacity in the DAT and DVDAT groups was significantly higher compared to the NC, DC, and DVD groups (p < 0.05). These findings suggest that type 2 diabetes is associated with excessive autophagy in the left ventricle. However, after eight weeks of vitamin D3 supplementation and aerobic training, a significant reduction in excessive autophagy was observed in rats with type 2 diabetes.

Autophagy inhibition mediated by trillin promotes apoptosis in hepatocellular carcinoma cells via activation of mTOR/STAT3 signaling.

Apoptosis and autophagy have been shown to act cooperatively and antagonistically in self-elimination process. On the one side, apoptosis and autophagy can act as partners to induce cell death in a coordinated or cooperative manner; on the flip side, autophagy acts as an antagonist to block apoptotic cell death by promoting cell survival. Our previous research indicated that trillin could induce apoptosis of PLC/PRF/5 cells, but the effects of trillin on autophagy as well as its functional relationship to apoptosis have not been elucidated. Here, the running study aims to investigate the function and molecular mechanism of trillin on autophagy with hepatocellular carcinoma (HCC) cells. The objective of this study is to investigate the molecular mechanism of trillin on autophagy in HCC cells. Protein levels of autophagy markers beclin1, LC3B, and p62 were detected by western blotting. 6-Hydroxyflavone and stattic were used to test the role of trillin regulation of autophagy via serine threonine kinase (AKT)/extracellular-regulated protein kinases (ERK) 1/2/mammalian target of rapamycin (mTOR)/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Flow cytometry was used to detect caspase 3 activity and apoptosis in PLC/PRF/5 cells treated with trillin for 24 h with or without rapamycin, stattic, and 6-hydroxyflavone. The protein level of autophagy marker beclin1 was decreased, whilst the protein level of p62 was significantly increased by trillin treatment, indicating trillin treatment led to inhibition of autophagy in HCC cells. Trillin treatment could reduce the protein levels of p-AKT and p-ERK1/2, but enhance the protein levels of mTOR and p-mTOR, suggesting that trillin could inhibit AKT/ERK rather than mTOR. The AKT/ERK activator 6-hydroxyflavone could reverse the loss of AKT and ERK1/2 phosphorylation induced by trillin, implying that trillin impairs autophagy through activated mTOR rather than AKT/ERK. STAT3 and p-STAT3 were significantly upregulated by the trillin treatment with an increase in dose from 0 to 50 μM, suggesting that autophagy inhibition is mediated by trillin via activation of STAT3 signaling. The STAT3 inhibitor stattic significantly reversed the increased STAT3 phosphorylation at tyrosine 705 induced by trillin. The mTOR signaling inhibitor rapamycin reversed the trillin-induced mTOR phosphorylation enhancement but exerted no effects on total mTOR levels, suggesting trillin treatment led to inhibition of autophagy in HCC cells through activating mTOR/STAT3 pathway. Furthermore, caspase 3 activities and the total rate of apoptosis were increased by trillin treatment, which was reversed by rapamycin, stattic, and 6-hydroxyflavone, proving that trillin promotes apoptosis via activation of mTOR/STAT3 signaling. Trillin induced autophagy inhibition and promoted apoptosis in PLC/PRF/5 cells via the activation of mTOR/STAT3 signaling. Trillin has the potential to be a viable therapeutic option for HCC treatment.

MiR-100-5p activation of the autophagy response through inhibiting the mTOR pathway and suppression of cerebral infarction progression in mice.

In recent years, the association between microRNAs (miRNAs) and autophagy in cerebral infarction (CI) has attracted increasingly more attention. The mammalian target of the rapamycin (mTOR) pathway is a key protein regulating the autophagy response. miR-100-5p can bind to the mTOR protein, but its role in CI remains unclear yet. This experiment aims to clarify the role of miR-100-5p in CI. Bioinformatics analysis was performed to screen differentiated expressed functional genes between CI tissue and normal tissue specimens. In vivo experiments: the mouse model of CI was established by middle cerebral artery occlusion (MCAO) methods, After being treated with miR-100-5p-overexpressing lentivirus, the amount of terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL)-positive fluorescence and the fluorescent expression level of mTOR protein were significantly inhibited in the CI region. Western blotting analysis showed that miR-100-5p inhibited the protein expression level of phosphorylated mTOR and total mTOR and enhanced the expression of autophagy-related proteins Beclin, microtubule-associated protein light chain 3II (LC-3II), and autophagy-related gene 7 (ATG-7). For in vitro experiment, after the BV-2 cells were successfully infected with the control lentivirus and miR-100-5p-overexpression lentivirus, they were stimulated with 1% hypoxia and low-glucose medium in a tri-gas incubator for 24 h. It was found that miR-100-5p could significantly lower the protein expression level of phosphorylated mTOR and total mTOR, and increase the expression of the Beclin, LC-3II, ATG-7 autophagy related proteins. miR-100-5p promotes the autophagy response through binding to mTOR protein, thereby inhibiting apoptosis and delaying the progression of CI.

Rhes depletion promotes striatal accumulation and aggregation of mutant huntingtin in a presymptomatic HD mouse model.

Huntington's disease (HD) is caused by CAG trinucleotide repeats in the HTT gene. Selective neurodegeneration in the striatum is prominent in HD, despite widespread expression of mutant HTT (mHTT). Ras homolog enriched in the striatum (Rhes) is a GTP-binding protein enriched in the striatum, involved in dopamine-related behaviors and autophagy regulation. Growing evidence suggests Rhes plays a critical role in the selective striatal degeneration in HD, but its specific function in this context remains complex and controversial. In this study, we utilized CRISPR/Cas9 to knockdown Rhes at different disease stages through adeno-associated virus (AAV) transduction in HD knock-in (KI) mice. Immunoblotting and immunofluorescence were employed to assess the impact of Rhes depletion on mHTT levels, neuronal loss, astrogliosis and autophagy activity. Rhes depletion in 22-week-old HD KI mice (representing the presymptomatic stage) led to mHTT accumulation, reduced neuronal cell staining, and increased astrogliosis. However, no such effects were observed in 36-week-old HD KI mice (representing the symptomatic stage). Additionally, Rhes deletion in 22-week-old HD KI mice resulted in increased P62 levels, reduced LC3-II levels, and unchanged phosphorylation of mTOR and beclin-1, unchanged mTOR protein level, except for a decrease in beclin-1. Our findings suggest that knockdown Rhes promotes striatal aggregation of mutant huntingtin by reducing autophagy activity in a mTOR-independent manner. Rhes plays a protective role during the presymptomatic stage of HD KI mice.

Effects of Xihuang Pills on proliferation and apoptosis of prostate cancer LNCaP cells based on AR/m TOR signaling pathway

Based on the androgen receptor(AR)/mammalian target of rapamycin(mTOR)signaling pathway, the effects of Xihuang Pills-medicated serum on the proliferation and apoptosis of prostate cancer LNCaP cells were investigated. The drug-containing serum of SD rats was prepared by intragastric administration of Xihuang Pills suspension. The effects of low-, medium-, and high-dose Xihuang Pills-containing serum on the in vitro proliferation of LNCaP cells were detected by cell counting kit-8(CCK-8). Flow cytometry was used to detect the apoptosis level of LNCaP cells after intervention with different concentrations of Xihuang Pills. Protein expression of cleaved cysteinyl aspartate-specific proteinase caspase-3(cleaved caspase-3), B-cell lymphoma-2(Bcl-2), and AR as well as the phosphorylation level of mTOR protein were detected by Western blot. The results showed that compared with the blank serum, the drug-medicated serum could blunt the activity of LNCaP cells. Low-, medium-, and high-dose Xihuang Pills-containing serum could significantly increase the cell apoptosis rate, increase the expression of cleaved caspase-3 protein, decrease the expression of Bcl-2 protein, reduce the expression of AR protein, and down-regulate the level of phosphorylated mTOR(p-mTOR). To study the effect of Xihuang Pills on the growth of LNCaP cells in vivo, different doses of Xihuang Pills were used to intervene in the subcutaneous graft model in nude mice inoculated with LNCaP cells. The expression levels of AR, mTOR, p-mTOR, Bcl-2, and cleaved caspase-3 were detected by Western blot. The results showed that the volumes of subcutaneous graft tumor in the low-dose, medium-dose, and high-dose Xihuang Pills groups significantly decreased compared with that in the model group. The weight of subcutaneous transplanted tumor in each group with drug intervention was significantly lower than that in the model group. Compared with the model group, the low-dose, medium-dose, and high-dose Xihuang Pills groups showed increased cleaved caspase-3 protein expression, decreased Bcl-2 and AR protein expression, and reduced p-mTOR protein expression. Further experiments showed that AR agonist R1881 could block the anti-proliferation and pro-apoptotic effects of Xihuang Pills. The mechanism of Xihuang Pills against prostate cancer is related to the inhibition of the AR/mTOR signaling pathway, inhibition of LNCaP cell proliferation, and induction of apoptosis in cancer cells.

Dietary oleic acid supplementation improves feed efficiency and modulates fatty acid profile and cell signaling pathway in European sea bass (Dicentrarchus labrax) juveniles fed high-lipid diets

Diets with high-lipid levels may reduce fish growth, feed intake and increase fat deposition, jeopardizing fillet quality and shelf-life, which may be of major concern for the final consumer. To mitigate these potentially harmful effects, several nutritional strategies have been applied, including the use of functional ingredients. In this study, European sea bass (initial body weight 21.4 g) were fed six isonitrogenous (45% crude protein) diets with 16% and 22% lipid levels and supplemented with oleic acid (OA) at 0, 1, and 2% as a functional ingredient.Increasing dietary lipid levels did not affect fish growth performance, feed intake, and feed utilization. Independently of lipid level, dietary OA supplementation decreased feed intake and increased feed efficiency and Nitrogen (N) retention (% N Intake). Whole-body composition, hepatosomatic and visceral somatic indexes were not affected by dietary composition. The liver and muscle lipid contents were also not affected by dietary composition.The liver fatty acid profile was not affected by dietary lipid level, but monounsaturated fatty acid (MUFA) content decreased while polyunsaturated fatty acid (PUFA) content decreased with dietary OA supplementation. In the muscle, PUFA content decreased with dietary lipid levels, particularly C18:2n-6 (LA) and C20:2n-6. Dietary OA supplementation increased muscle MUFA, particularly OA (C18:1n-9), and reduced PUFA content. Compared to fish fed with 1OA diets, fish fed with 2OA diets presented a decreased area covered by lipids in the liver.Dietary OA supplementation increased hepatic glucose-6-phosphate dehydrogenase (G6PDH) activity and decreased malic enzyme (ME) activity, while no differences were observed in the activity of fatty acid synthase (FAS) and enzymes related to β-oxidation. Moreover, dietary OA supplementation decreased mechanistic target of rapamycin (mTOR) protein levels, while protein kinase B (Akt) and the phosphorylated state levels of both Akt and mTOR were not affected by dietary composition. Overall, dietary OA supplementation decreased feed intake and improved feed efficiency, without affecting growth performance, contributing to mitigate the potentially harmful effects of high-lipid diets in European sea bass.

The effect of different doses of nonylphenol on the blood-testicular barrier integrity, hormone level, and DNA damage in the testes of rats.

Determining the molecular characteristics of the damage caused by NP exposure in the testis is very important for understanding the source of the damage and developing treatment methods accordingly. Therefore, in this study, it is aimed to evaluate the toxic effects that different doses of NP may cause in the testis, including blood-testicular barrier integrity and sperm DNA damage. For this purpose, 50 adult male Wistar albino rats were used in the study. Low, medium, and high-dose NP groups and the corn oil group were formed. After NP administration at determined doses for 15 days, the testis tissue taken under anesthesia was fixed in formaldehyde. Paraffin blocks were embedded using the routine histological tissue follow-up method. Histopathological and immunohistochemical analyses were performed by taking 5μm thick sections from paraffin blocks. The other testicular tissue was taken for the Western blot, Elisa, and comet methods, and the findings of sperm DNA analysis and the blood-testicular barrier were examined. NP caused the seminiferous epithelium to be disorganized and have significantly fewer cells in the testes of rats in different dose NP-induced groups. Compared with the control group, mTOR, Cx43, SCF, and HSP70 protein levels were decreased, while the expression of MMP-9 levels was increased in the different NP dose groups. Furthermore, tissue testosterone and inhibin B levels and SF-1 immunoreactivity intensity gradually decreased depending on the dose increase of NP. DNA damage of testicular tissues were increased in NP groups depending on NP dose. These results suggest that it is evident that NP, a commonly used industrial chemical, is an endocrine disrupting chemical (EDC) with estrogenic activity exerting adverse effects on health and that urgent measures are needed regarding the use.

Maternal diabetes increases FOXO1 activation during embryonic cardiac development

Maternal diabetes is known to affect heart development, inducing the programming of cardiac alterations in the offspring's adult life. Previous studies in the heart of adult offspring have shown increased activation of FOXO1 (a transcription factor involved in a wide variety of cellular functions such as apoptosis, cellular proliferation, reactive oxygen species detoxification, and antioxidant and pro-inflammatory processes) and of target genes related to inflammatory and fibrotic processes. In this work, we aimed to evaluate the effects of maternal diabetes on FOXO1 activation as well as on the expression of target genes relevant to the formation of the cardiovascular system during organogenesis (day 12 of gestation). The embryonic heart from diabetic rats showed increased active FOXO1 levels, reduced protein levels of mTOR (a nutrient sensor regulating cell growth, proliferation and metabolism) and reduced mTORC2-SGK1 pathway, which phosphorylates FOXO1. These alterations were related to increases in the levels of 4-hydroxynonenal (an oxidative stress marker) and increased mRNA levels of inducible nitric oxide synthase, angiopoietin-2 and matrix metalloproteinase-2 (MMP2) (all FOXO1 target genes relevant for cardiac development). Results also showed increased extracellular and intracellular immunolocalization of MMP2 in the myocardium and its projection into the lumen of the cavity (trabeculations) together with decreased immunostaining of connexin 43, a protein relevant for cardiac function that is target of MMP2. In conclusion, increases in active FOXO1 induced by maternal diabetes initiate early during embryonic heart development and are related to increases in markers of oxidative stress and of proinflammatory cardiac development, as well to an altered expression of proteolytic enzymes that regulate connexin 43. These alterations may lead to an altered programming of cardiovascular development in the embryonic heart of diabetic rats.

Shaofu Zhuyu Decoction attenuates fibrosis in endometriosis through regulating PTEN/Akt/mTOR signaling pathway

The present study aimed to investigate the protective role of Shaofu Zhuyu Decoction(SFZY) against endometriosis fibrosis in mice, and decipher the underlying mechanism through the phosphatase and tensin homolog deleted on chromosome ten(PTEN)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR) pathway. Eighty-five BALB/c female mice were randomly assigned into a blank group, a model group, high-, medium, and low-dose SFZY(SFZY-H, SFZY-M, and SFZY-L, respectively) groups, and a gestrinone suspension(YT) group. The model of endometriosis was induced by intraperitoneal injection of uterine fragments. The mice in different groups were administrated with corresponding groups by gavage 14 days after modeling, and the blank group and model group with equal volume of distilled water by gavage. The treatment lasted for 14 days. The body weight, paw withdrawal latency caused by heat stimuli, and total weight of dissected ectopic focus were compared between different groups. The pathological changes of the ectopic tissue were observed via hematoxylin-eosin(HE) and Masson staining. Real-time PCR was employed to measure the mRNA levels of α-smooth muscle actin(α-SMA) and collagen type Ⅰ(collagen-Ⅰ) in the ectopic tissue. The protein levels of PTEN, Akt, mTOR, p-Akt, and p-mTOR in the ectopic tissue were determined by Western blot. Compared with the blank group, the modeling first decreased and then increased the body weight of mice, increased the total weight of ectopic focus, and shortened the paw withdrawal latency. Compared with the model group, SFZY and YT increased the body weight, prolonged the paw withdrawal latency, and decreased the weight of ectopic focus. Furthermore, the drug administration, especially SFZY-H and YT(P&lt;0.01), recovered the pathological and reduced the area of collagen deposition. Compared with the blank group, the modeling up-regulated the mRNA levels of α-SMA and collagen-Ⅰ in the ectopic focus, and such up-regulation was attenuated after drug intervention, especially in the SFZY-H and YT groups(P&lt;0.05,P&lt;0.01). Compared with the blank group, the modeling down-regulated the protein level of PTEN and up-regulated the protein levels of Akt, mTOR, p-Akt, and p-mTOR(P&lt;0.01, P&lt;0.001). Drug administration, especially SFZY-H and YT, restored such changes(P&lt;0.01). SFZY may significantly attenuate the focal fibrosis in the mouse model of endometriosis by regulating the PTEN/Akt/mTOR signaling pathway.

Periplaneta americana extract CⅡ-3 induces senescence of leukemia K562 cells via SIRT1/mTOR signaling pathway

This study aims to investigate the role of slient mating-type information regulation 2 homolog 1(SIRT1)/tuberous sclerosis complex 2(TSC2)/mammalian target of rapamycin(mTOR) signaling pathways in the Periplaneta americana extract CⅡ-3-induced senescence of human leukemia K562 cells. K562 cells were cultured in vitro and treated with 0(control), 5, 10, 20, 40, 80, and 160 μg·mL~(-1) of P. americana extract CⅡ-3. Cell counting kit-8(CCK-8) and flow cytometry were employed to examine the proliferation and cell cycle of the K562 cells. Senescence-associated β-galactosidase stain kit(SA-β-gal) was used to detect the positive rate of senescent cells. Mitochondrial membrane potential was detected by flow cytometry. The relative mRNA level of telomerase reverse transcriptase(TERT) was determined by fluorescence quantitative PCR. The mRNA and protein levels of SIRT1, TSC2, and mTOR were determined by fluorescence quantitative PCR and Western blot, respectively. The results showed that CⅡ-3 significantly inhibited the proliferation of K562 cells and the treatment with 80 μg·mL~(-1) CⅡ-3 for 72 h had the highest inhibition rate. Therefore, 80 μg·mL~(-1) CⅡ-3 treatment for 72 h was selected as the standard for subsequent experiments. Compared with the control group, CⅡ-3 increased the proportion of cells arrested in G_0/G_1 phase, decreased the proportion of cells in S phase, increased the positive rate of SA-β-Gal staining, elevated the mitochondrial membrane potential and down-regulated the mRNA expression of TERT. Furthermore, the mRNA expression of SIRT1 and TSC2 was down-regulated, while the mRNA expression of mTOR was up-regulated. The protein expression of SIRT1 and p-TSC2 was down-regulated, while the protein expression of p-mTOR was up-regulated. The results indicated that P. americana extract CⅡ-3 induced the senescence of K562 cells via the SIRT1/mTOR signaling pathway.

Downregulation of BRD4 attenuates high glucose-induced damage of trophoblast cells by inhibiting activation of AKT/mTOR pathway.

It was elucidated that bromodomain-containing protein 4 (BRD4) has involvement with diabetic complication. However, the role and molecular mechanism of BRD4 in gestational diabetes mellitus (GDM) are still unclear. In this study, the mRNA and protein contents of BRD4 in placenta tissues of GDM patients and high glucose (HG)-induced HTR8/SVneo cells were detected by qRT-PCR and western blot assay. CCK-8, EdU staining, flow cytometry as well as western blot were applied for the appraisement of cell viability and apoptosis. Wound healing assay and transwell assay were conducted for the assessment of cell migration and invasion. Oxidative stress and inflammatory factors were detected. Additionally, the contents of AKT/mTOR pathway-related proteins were estimated applying western blot. It was discovered that BRD4 expression was ascended in tissues and HG-induced HTR8/SVneo cells. BRD4 downregulation cut down the contents of p-AKT and p-mTOR but had no effects on the total protein levels of AKT or mTOR in HG-induced HTR8/SVneo cells. BRD4 depletion promoted cell viability, enhanced proliferative capability, and reduced cell apoptotic level. Moreover, BRD4 depletion facilitated cell migrative and invasive capabilities, and repressed the oxidative stress as well as inflammatory damage in HG-induced HTR8/SVneo cells. The activation of Akt reversed the protective impacts of BRD4 depletion on HG-induced HTR8/SVneo cells. To sum up, BRD4 silencing may alleviate HG-induced HTR8/SVneo cell damage through the inhibition of the AKT/mTOR pathway.

Wip1 regulates wound healing by affecting activities of keratinocytes and endothelial cells through ATM-p53 and mTOR signaling

BackgroundAs a p53-regulated gene, Wip1 regulates proliferation, migration, apoptosis, and senescence of several type cells, but its biological functions in keratinocytes and endothelial cells which are involved wound healing are not fully understood. This study aims to reveal the function and underlying mechanism of Wip1 in wound healing using models of transgenic animal, keratinocytes, and endothelial cells. MethodsUsing Wip1 knockout C57 BL/6 mice, we investigated effect of Wip1 deficiency on wound healing and angiogenesis; And using HaCaT and HUVEC as keratinocytes and endothelial cells, combined using primary keratinocytes from Wip1 knockout mice, we studied the effects of Wip1 knockdown/knockout or overexpression on proliferation, migration, and protein expressions of signaling components in ATM-p53 and mTOR pathway. ResultsWip1 deficiency in mice impaired the wound repair and endothelial angiogenesis, reduced the thickness of granulation tissue, and decreased the number of Ki67-positive cells and CD31 positive vessels in granulation tissue. Knockdown of Wip1 by shRNAs suppressed the proliferation and migration of HaCaT and HUVEC cells and induced notably apoptosis in the two cells. In western blot, Wip1 knockdown enriched p53 and ATM proteins, while decreased activated AKT, mTOR and activated S6 ribosomal protein (pS6) levels in HaCaT and HUVEC cells. Ectopic expression of Wip1 decreased the p53 and ATM proteins, while increased activated AKT, mTOR and pS6 levels in HaCaT and HUVEC cells. And in primary keratinocytes from mice tail skin, Wip1 knockout increased p53 and ATM, while decreased activated AKT, mTOR and pS6 protein levels. ConclusionOur study directly supports that Wip1 regulated skin wound healing possibly by affecting bioactivities including proliferation, migration and apoptosis of keratinocytes and endothelial cells at least through by modulating ATM-p53 and mTOR signaling.

Circadian regulation of mTORC1 signaling via Per2 dependent mechanism disrupts folliculogenesis and oocyte maturation in female mice.

mTOR (mammalian target of Rapamycin) is an important signaling pathway involved in several crucial ovarian functions including folliculogenesis and oocyte maturation. The circadian rhythm regulates multiple physiological processes and PER2 is one of the core circadian rhythm components. mTOR is regulated by the circadian clock and in turn, the rhythmic mTOR activities strengthen the clock function. Our current study aims to investigate a possible interconnection between the circadian clock and the mTORC1 signaling pathway in folliculogenesis and oocyte maturation. Here we demonstrate that the circadian system regulates mTORC1 signaling via Per2 dependent mechanism in the mouse ovary. To investigate the effect of constant light on ovarian and oocyte morphology, animals were housed 12:12h L:D group in standard lightening conditions and the 12:12h L:L group in constant light for one week. Food intake and body weight changes were measured. Ovarian morphology, follicle counting, and oocyte aging were evaluated. Afterward, western blot for mTOR, p-mTOR, p70S6K, p-p70S6K, PER2, and Caspase-3 protein levels was performed. The study demonstrated that circadian rhythm disruption caused an alteration in their food intake and decrease in primordial follicle numbers and an increase in the number of atretic follicles. It caused an increase in oxidative stress and a decrease in ZP3 expression in oocytes. Decreased protein levels of mTOR, p-mTOR, p70S6K, and PER2 were shown. The results showed that the circadian clock regulates mTORC1 through PER2 dependent mechanism and that decreased mTORC1 activity can contribute to premature aging of mouse ovary. In conclusion, these results suggest that the circadian clock may control ovarian aging by regulating mTOR signaling pathway through Per2 expression.

Psilocybin facilitates fear extinction in mice by promoting hippocampal neuroplasticity.

Posttraumatic stress disorder (PTSD) and depression are highly comorbid. Psilocybin exerts substantial therapeutic effects on depression by promoting neuroplasticity. Fear extinction is a key process in the mechanism of first-line exposure-based therapies for PTSD. We hypothesized that psilocybin would facilitate fear extinction by promoting hippocampal neuroplasticity. First, we assessed the effects of psilocybin on percentage of freezing time in an auditory cued fear conditioning (FC) and fear extinction paradigm in mice. Psilocybin was administered 30 min before extinction training. Fear extinction testing was performed on the first day; fear extinction retrieval and fear renewal were tested on the sixth and seventh days, respectively. Furthermore, we verified the effect of psilocybin on hippocampal neuroplasticity using Golgi staining for the dendritic complexity and spine density, Western blotting for the protein levels of brain derived neurotrophic factor (BDNF) and mechanistic target of rapamycin (mTOR), and immunofluorescence staining for the numbers of doublecortin (DCX)- and bromodeoxyuridine (BrdU)-positive cells. A single dose of psilocybin (2.5 mg/kg, i.p.) reduced the increase in the percentage of freezing time induced by FC at 24 h, 6th day and 7th day after administration. In terms of structural neuroplasticity, psilocybin rescued the decrease in hippocampal dendritic complexity and spine density induced by FC; in terms of neuroplasticity related proteins, psilocybin rescued the decrease in the protein levels of hippocampal BDNF and mTOR induced by FC; in terms of neurogenesis, psilocybin rescued the decrease in the numbers of DCX- and BrdU-positive cells in the hippocampal dentate gyrus induced by FC. A single dose of psilocybin facilitated rapid and sustained fear extinction; this effect might be partially mediated by the promotion of hippocampal neuroplasticity. This study indicates that psilocybin may be a useful adjunct to exposure-based therapies for PTSD and other mental disorders characterized by failure of fear extinction.

Branched-Chain Amino Acids and Di-Alanine Supplementation in Aged Mice: A Translational Study on Sarcopenia.

In age-related sarcopenia, the gradual loss of skeletal muscle mass, function and strength is underpinned by an imbalanced rate of protein synthesis/breakdown. Hence, an adequate protein intake is considered a valuable strategy to mitigate sarcopenia. Here, we investigated the effects of a 12-week oral supplementation with branched-chain amino acids (BCAAs: leucine, isoleucine, and valine) with recognized anabolic properties, in 17-month-old (AGED) C57BL/6J male mice. BCAAs (2:1:1) were formulated in drinking water, alone or plus two L-Alanine equivalents (2ALA) or dipeptide L-Alanyl-L-Alanine (Di-ALA) to boost BCAAs bioavailability. Outcomes were evaluated on in/ex vivo readouts vs. 6-month-old (ADULT) mice. In vivo hind limb plantar flexor torque was improved in AGED mice treated with BCAAs + Di-ALA or 2ALA (recovery score, R.S., towards ADULT: ≥20%), and all mixtures significantly increased hind limb volume. Ex vivo, myofiber cross-sectional areas were higher in gastrocnemius (GC) and soleus (SOL) muscles from treated mice (R.S. ≥ 69%). Contractile indices of isolated muscles were improved by the mixtures, especially in SOL muscle (R.S. ≥ 20%). The latter displayed higher mTOR protein levels in mice supplemented with 2ALA/Di-ALA-enriched mixtures (R.S. ≥ 65%). Overall, these findings support the usefulness of BCAAs-based supplements in sarcopenia, particularly as innovative formulations potentiating BCAAs bioavailability and effects.

Unleashing lactoferrin's antidepressant potential through the PI3K/Akt/mTOR pathway in chronic restraint stress rats.

Depression is a widespread neuropsychiatric illness whose etiology is yet mysterious. Lactoferrin (LF), an iron-binding glycoprotein, is reported to promote neuroprotection through its role in the modulation of oxidative stress and inflammation. The objective of the present research was to evaluate the efficacy of LF against chronic restraint stress (CRS)-induced depressive behavior in rats. Depression was evidenced by a reduced grooming time in the splash test and an increased immobility time in the tail suspension test (TST) and forced swimming test (FST). This effect was also accompanied by reduced GSH and serotonin levels and elevated lipid peroxidation and corticosterone levels in the hippocampus. Additionally, an exaggerated hippocampal inflammatory response was also shown by a rise in NF-κB (p65) and TNF-α levels and a reduced IL-10 level. Moreover, CRS substantially reduced the BDNF content as well as the protein levels of PI3K, Akt, and mTOR while boosting the GSK3β content. Interestingly, LF therapy significantly improved CRS-induced behavioral and biochemical aberrations, an effect which was suppressed upon pretreatment with LY294002 (PI3K inhibitor). This suggests that the antidepressant potential of LF may be mediated through the modulation of the PI3K/Akt/mTOR signaling pathway. Furthermore, LF succeeded in restoring 5-HT and corticosterone levels, diminishing oxidative stress and ameliorating the inflammatory cascades. Therefore, and for the first time, LF might serve as a promising antidepressant drug through targeting the PI3K/Akt/mTOR pathway.

Evaluation the cognition-improvement effects of N-acetyl cysteine in experimental temporal lobe epilepsy in rat

Memory impairment is a critical issue in patients with temporal lobe epilepsy (TLE). Neuronal loss within the hippocampus and recurrent seizures may cause cognitive impairment in TLE. N -acetyl cysteine (NAC) is a sulfur-containing amino acid cysteine that is currently being investigated due to its protective effects on neurodegenerative disorders. NAC was orally administrated at a dose of 100 mg/kg for 8 days (7-day pretreatment and 1-day post-surgery). Neuronal viability, mTOR protein level, and spatial memory were detected in the kainite temporal epilepsy model via Nissl staining, western blot method, and Morris water maze task, respectively. Results showed that NAC delayed seizure activity and ameliorated memory deficit induced by Kainic acid. Histological analysis showed that NAC significantly increased the number of intact neurons in CA3 and hilar areas of the hippocampus following the induction of epilepsy. NAC also modulated the mTOR protein level 5 days after epilepsy compared to the KA-induced group. ConclusionThese results suggest that NAC improved memory impairment via anticonvulsant and neuroprotective activity and, in all probability, by lowering the level of mTOR.