mRNA Expression Of mTOR Research Articles

Effect of needle knife on mTOR/Atg/ULK1/Beclin-1 axis and chondrocyte autophagy in rats with knee osteoarthritis

To observe the effect of needle knife on chondrocyte autophagy and expressions of autophagy-related protein and mammalian target of rapamycin (mTOR) in rats with knee osteoarthritis (KOA), and to explore the possible mechanism of needle knife for KOA. A total of 42 SD rats were randomly divided into a normal group, a model group and a needle knife group, 14 rats in each group. Except for the normal group, the other two groups were injected with the mixture of papain and L-cysteine into the left hind knee joint to establish the KOA model. After modeling, the rats in the needle knife group were treated with needle knife at strip or nodule around the quadriceps femoris and medial and lateral collateral ligament on the affected side, once a week for 3 times (3 weeks). The changes of left knee circumference in each group were observed; the chondrocytes and ultrastructure of left knee joint were observed by HE staining and electron microscope; the mRNA and protein expressions of autophagy-related genes (Atg5, Atg12, Atg4a), Unc-51 like autophagy activated kinase 1 (ULK1), autophagy gene Beclin-1 and mTOR in left knee cartilage were detected by real-time fluorescence quantitative PCR and Western blot. After modeling, the left knee circumferences in the model group and the needle knife group were increased compared with those before modeling and in the normal group (P<0.05); after intervention, the left knee circumference in the needle knife group was smaller than that in the model group and after modeling (P<0.05). Compared with the normal group, the number of chondrocytes was decreased, and a few cells swelled, nuclei shrank, mitochondria swelled and autophagosomes decreased in the model group; compared with the model group, the number of chondrocytes was increased , and most cell structures returned to normal, and autophagosomes was increased. Compared with the normal group, the mRNA and protein expressions of Atg5, Atg12, Atg4a, Beclin-1 and ULK1 in the knee cartilage in the model group were decreased (P<0.05); compared with the model group, the expressions of the above indexes in the needle knife group were increased (P<0.05). Compared with the normal group, the mRNA and protein expressions of mTOR in the knee cartilage in the model group were increased (P<0.05); compared with the model group, the expressions of the above indexes in the needle knife group were decreased (P<0.05). The needle knife intervention could improve knee cartilage injury in rats with KOA, and its mechanism may be related to reducing the expression of mTOR and up-regulating the expressions of Atg5, Atg12, Atg4a, ULK1 and Beclin-1, so as to promote chondrocyte autophagy and delay the aging and degeneration of chondrocytes.

Roles of pulmonary telocytes in airway epithelia to benefit experimental acute lung injury through production of telocyte-driven mediators and exosomes.

BackgroundTelocytes (TCs) are experimentally evidenced as an alternative of cell therapies for organ tissue injury and repair. The aims of the present studies are to explore direct roles of TCs and the roles of TC-derived exosomes in support of experimental acute lung injury (ALI) in vivo or in vitro.Materials and methodsThe roles of TCs in experimental ALI were firstly estimated. Phosphoinositide 3-kinase (PI3K) p110δ and α/δ/β isoform inhibitors were used in study dynamic alterations of bio-behaviors, and in expression of functional factors of TCs per se and TC-co-cultured airway epithelial cells during the activation with lipopolysaccharide (LPS). TC-driven exosomes were furthermore characterized for intercellular communication by which activated or non-activated TCs interacted with epithelia. ResultsOur results showed that TCs mainly prevented from lung tissue edema and hemorrhage and decreased the levels of VEGF-A and MMP9 induced by LPS. Treatment with CAL101 (PI3K p110δ inhibitor) and LY294002 (PI3Kα/δ/β inhibitor) could inhibit TC movement and differentiation and increase the number of dead TCs. The expression of Mtor, Hif1α, Vegf-a, or Mmp9 mRNA increased in TCs challenged with LPS, while Mtor, Hif1α, and Vegf-a even more increased after adding CAL101 or Mtor after adding LY. The rate of epithelial cell proliferation was higher in co-culture of human bronchial epithelial (HBE) and TCs than that in HBE alone under conditions with or without LPS challenge or when cells were treated with LPS and CAL101 or LY294002. The levels of mTOR, HIF1α, or VEGF-A significantly increased in mono-cultured or co-cultured cells, challenged with LPS as compared with those with vehicle. LPS-pretreated TC-derived exosomes upregulated the expression of AKT, p-AKT, HIF1α, and VEGF-A protein of HBE.ConclusionThe present study demonstrated that intraperitoneal administration of TCs ameliorated the severity of lung tissue edema accompanied by elevated expression of VEGF-A. TCs could nourish airway epithelial cells through nutrients produced from TCs, increasing epithelial cell proliferation, and differentiation as well as cell sensitivity to LPS challenge and PI3K p110δ and α/δ/β inhibitors, partially through exosomes released from TCs.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s10565-021-09670-5.

Rehmanniae Radix-Induced apoptosis via inhibition of PI3K/AKT/mTOR signaling pathways in human hepatocellular carcinoma cell lines SMMC-7721

Background: Hepatocellular carcinoma (HCC) grades second among cancer-related deaths. Rehmanniae Radix (RR) has been exposed to have numerous pharmacological properties. Objectives: To inspect RR leaves extracts pro-apoptotic activities on SMMC-7721 liver cancer cell lines. Materials and Methods: Anti-cancer effect of RR extracts in SMMC-7721 liver cancer cell lines was aged rate of cell apoptosis (AO/EB, 4′,6-diamidino-2-phenylindole [DAPI], and PI staining), ΔΨm (Rh-123 staining), intracellular Reactive oxygen species (ROS) (DCFH-DA staining), quantitative polymerase chain reaction (mRNA expressions of Bax, p53, Bcl-2, caspase-3,-9,-8, IL-6, PI3K/AKT, and mTOR). Results: It has been shown that RR leaf ethanolic extracts suggestively declined cell viability, augmented the rate of cell apoptosis (AO/EB, DAPI, and PI staining), and caused the potential for ΔΨm, and induced over-accumulation of intracellular ROS (DCFH-DA staining) in SMMC-7721 cells after 24 h of exposure. In SMMC-7721 cell lines, we have examined anti-cancer activity to scrutinize the underlying mechanism by assessing the mRNA expressions of Bax, p53, Bcl-2, caspase-3,-9,-8, IL-6, PI3K/AKT, and mTOR. Results displayed the significant inhibition of the apoptosis as stated above related proteins. Conclusion: RR extracts exhibited its potential anti-cancer activity against SMMC-7721 cells by prompting apoptosis-induced cell death and inhibiting PI3K/AKT/mTOR signaling.

The Chemosensitizing Effect of Portulaca oleracea Methanol Extract on Human Colorectal and Lung Cancer Cells Against Irinotecan

Cancer is a major health problem and it has been causing great damage in recent years worldwide. Serious side effects of chemotherapeutics used in cancer treatment make natural products valuable. Combined use of chemotherapy drugs and herbal products is an important approach in reducing side effects and revealing a more effective treatment. In this study, the effects of methanolic extract from Portulaca oleracea (POM) alone and in combination with Irinotecan were investigated against human colorectal carcinoma cell line HCT-116 and human lung adenocarcinoma cell line A549. The antiproliferative/anti-colony formation effects were determined using WST-1 assay and clonogenic assay. Cell death detection Elisa assay was employed to exhibite the triggered apoptosis, and quantitative real time polimerase chain reaction (qRT-PCR) was performed to evaluate the mRNA expression of mTOR, Akt and PTEN genes, which are significant modulators of apoptotic and survival pathways. It was determined that 72 h treatment with increasing concentrations of POM (0-1000 mg mL-1) decreased the viability of HCT116 and A549 cells in a dose-dependent manner. Also, POM and Irinotecan exhibited synergistic effects in both cell lines. The combination of POM and Irinotecan suppressed the colony forming capacity of HCT116 and A549 cells by 84% and 79%, respectively. POM also altered mTOR, Akt and PTEN gene expressions in HCT-116 and A549 carcinoma cells, both alone and in combination with Irinotecan. This study shows that the phytotherapeutic effect of P. oleracea needs to be studied more intensively.

Taurine stimulates protein synthesis and proliferation of C2C12 myoblast cells through the PI3K-ARID4B-mTOR pathway.

Taurine (Tau) has many profound physiological functions, but its role and molecular mechanism in muscle cells are still not fully understood. In this study, we investigated the role and underlying molecular mechanism of Tau on protein synthesis and proliferation of C2C12 myoblast cells. Cells were treated with Tau (0, 60, 120, 180 and 240 μM) for 24 h. Tau dose-dependently promoted protein synthesis, cell proliferation, mechanistic target of rapamycin protein (mTOR) phosphorylation and also AT-rich interaction domain 4B (ARID4B) expression, with the best stimulatory effects at 120 μM. LY 294002 treatment showed that Tau promoted ARID4B expression in a phosphoinositide 3-kinase (PI3K)-dependent manner. ARID4B knockdown (by small interfering RNA transfection for 24 h) prevented Tau from stimulating protein synthesis and cell proliferation, whereas ARID4B gene activation (using the CRISPR/dCas9 technology) had stimulatory effects. ARID4B knockdown abolished Tau signalling to mRNA expression and protein phosphorylation of mTOR, whereas ARID4B gene activation had stimulatory effects. Chromatin immunoprecipitation (ChIP)-PCR identified that all of ARID4B, H3K27ac and H3K27me3 bound to the -4368 to -4591 bp site in the mTOR promoter, and ChIP-quantitative PCR (qPCR) further detected that Tau stimulated ARID4B binding to this site. ARID4B knockdown or gene activation did not affect H3K27me3 binding to the mTOR promoter but decreased or increased H3K27ac binding, respectively. Furthermore, ARID4B knockdown abolished the stimulation of Tau on H3K27ac binding to the mTOR promoter. In summary, these data uncover that Tau promotes protein synthesis and proliferation of C2C12 myoblast cells through the PI3K-ARID4B-mTOR pathway, providing a deep understanding of how Tau regulates anabolism in muscle cells.

Effecacy of Biejia (Carapax Trionycis) and Ezhu (Rhizoma Curcumae Phaeocaulis) couplet medicine on epithelial-mesenchymal transition, invasion and migration of MDA-MB-231 triple negative breast cancer cells via PI3K/Akt/mTOR signaling pathway.

To investigate the efficacy of Biejia (Carapax Trionycis) and Ezhu (Rhizoma Curcumae Phaeocaulis) couplet medicine on epithelial-mesenchymal transition (EMT), invasion and migration of MDA-MB-231 triple negative breast cancer (TNBC) cells based on PI3K/Akt/mTOR signaling pathway. MDA-MB-231 cells were treated with different medicated serum as Biejia-, Ezhu-, Biejia-Ezhu (BJ-, EZ-, BJ-EZ-) groups, intervened with no drug rat serum and paclitaxel with final concentration of 33 nM (IC50) as negative and positive control (NC and PC) groups. CCK-8 assay, scratch test, and Transwell assay were used to examine cell proliferation, invasion, and migration. The expression of E-cadherin, N-cadherin, Vimentin, MMP-2, MMP-9, PI3K, Akt, p-Akt, mTOR, and p-mTOR was determined by Western blot, and the mRNA expression of PI3K, Akt and mTOR was determined by real-time polymerase chain reaction. BJ-EZ group inhibited proliferation after 24, 48, and 72 h compared with the NC group (P < 0.05, < 0.01 or < 0.001) and reduced the invasion and migration of MDA-MB-231 cells (P < 0.01 or < 0.001). In addition, BJ-EZ group upregulated the expression of E-cadherin, downregulated the expression of N-cadherin, Vimentin, MMP-2, and MMP-9 (P < 0.05, P < 0.01 or P < 0.001), and inhibited the mRNA and protein expression of PI3K, Akt (p-Akt), mTOR (p-mTOR) (P < 0.05, < 0.01 or < 0.001). Biejia (Carapax Trionycis) and Ezhu (Rhizoma Curcumae Phaeocaulis) couplet medicine can inhibit the proliferation, invasion, migration and EMT of MDA-MB-231 cells through PI3K/Akt/mTOR signaling pathway, and the effect is better than that of Biejia (Carapax Trionycis) or Ezhu (Rhizoma Curcumae Phaeocaulis) alone.

Placental Insulin-Like Growth Factor-1 Receptor, Protein Kinase B and Mammalian Target of Rapamycin are Downregulated in HIV-1 Positive Women on Antiretroviral Drugs

HIV-1 and ARV drugs uptake during pregnancy may change placental phenotype during pregnancy affecting fetal growth. We investigated the influence of maternal HIV-1 and ARV drugs on expression of placental genes important for fetus growth. A total 51 HIV-1 positives and 46 HIV-1 negative pregnant women were studied. Placental gene expression changes of insulin-like growth factor receptor 1 (IGF-1) R, mammalian target of rapamycin (mTOR), protein kinase B (AKT-1), sodium-coupled neutral amino acid transporters (Slc38a1, Slca38a2, Slc38a4), inhibin A, adrenomedullin and 11 beta-Hydroxysteroid dehydrogenase type (HSD)-2 were assessed by RT-qPCR. There was a significant decrease in mRNA expression of placental IGF-1R, mTOR, and AKT-1 in HIV-1 positive placentas compared to controls (p &lt; 0.0001). There was also significant upregulation of an antiangiogenic molecule, inhibin A and downregulation of angiogenic molecule adrenomedullin in HIV-1 positive placenta (all p &lt; 0.0001). However, the mRNA expression of placental Slc38a1 and Slca38a2 was higher in both HIV-1 positive and negative women delivering LBW babies compared to controls (p &lt; 0.0001). The placental mRNA expression of 11 β-HSD-2 increased by 17 folds in HIV-1 negative and by 3.8 folds in HIV-1 positive women delivering LBW babies. IGF-1-P13-AKT-1-mTOR signaling pathway is dysregulated in placenta of HIV-1 positive women on ARV drugs. Higher mRNA expression levels of inhibin A and lower levels of adrenomedullin occur in placenta of HIV-1 positive women delivering LBW babies. ARV drugs and HIV-1 may be involved in the disruption of vascular tone of the placenta and therefore placental perfusion.&#x0D; Keywords: Pregnant women, HIV-1, ARV drugs, Placental IGF-1-AKT-1-mTOR signaling, inhibin A, adrenomedullin, amino acid transporters.

Methionine and leucine induce ARID1A degradation to promote mTOR expression and milk synthesis in mammary epithelial cells

Amino acids can activate mTOR to promote milk synthesis in mammary epithelial cells (MECs), but the underlying molecular mechanism is still largely unknown. The objective is to investigate the regulatory mechanism of amino acids (Met and Leu) in stimulating mRNA expression of mTOR in MECs. We found that the protein abundance of AT-rich interaction domain 1A (ARID1A) was poorly expressed in mouse mammary gland tissues of lactating period. ARID1A knockdown and gene activation experiments detected whether ARID1A negatively regulated milk protein and fat synthesis in bovine MECs, cell proliferation and the expression and activation of mTOR. ChIP-PCR detected that ARID1A, H3K27ac, H3K27me3 and H3K4me3 all bound to the mTOR promoter at -548∼-793 nt. Knockdown or gene activation of ARID1A enhanced or weakened the binding of H3K27ac on the mTOR promoter, respectively. The stimulation of Met and Leu on mTOR expression and phosphorylation were eliminated by ARID1A gene activation. Furthermore, Met and Leu decreased the protein level of ARID1A through ubiquitination and proteasomal degradation. TRIM21 bound to ARID1A, and TRIM21 knockdown blocked the stimulation of Met and Leu on ARID1A degradation. In summary, these data reveal that ARID1A blocks Met and Leu signaling to mTOR gene transcription through inhibiting H3K27ac deposition on its promoter, and Met and Leu decrease ARID1A protein level through TRIM21-mediated ubiquitination and proteasomal degradation. Our findings uncover that Met and Leu promote mTOR expression for milk synthesis through the TRIM21-ARID1A signaling pathway, providing a novel theoretical basis for the application of amino acids in milk production.

The regulatory effects of clomiphene and tamoxifen on mTOR and LC3-II expressions in relation to autophagy in experimental polycystic ovary syndrome (PCOS).

Polycystic ovary syndrome (PCOS) is a metabolic disease that causes infertility due to anovulation in women in reproductive age. It is known that clomiphene citrate (CC) and tamoxifen citrate (TMX) induce ovulation in women with PCOS. In this study, we aimed to investigate the effects of CC and TMX on the autophagy pathway in PCOS. Experimental PCOS model was induced by letrozole (1mg/kg) in rats by gavage for 21days. After the last letrozole administration, rats were treated TMX (1mg/kg) or CC (1mg/kg) for 5days. At the end of the experimental procedures, rats in all groups were sacrificed and ovarian tissues were removed. It was observed that mRNA and protein expressions of LC3-II were significantly higher in TMX and CC groups than control and PCOS groups (p < 0.05), while mRNA and protein expressions of mTOR in TMX and CC groups were found significantly lower than control and PCOS groups (p < 0.05). In conclusion, present study suggests that TMX and CC induce autophagy in ovaries with PCOS. Autophagy is a promising target for understanding pathophysiology of this disease and for developing more effective and safe new protocols for the treatment of PCOS-related anovulation.

Effect of Heat Stress on Bovine Mammary Cellular Metabolites and Gene Transcription Related to Amino Acid Metabolism, Amino Acid Transportation and Mammalian Target of Rapamycin (mTOR) Signaling.

Simple SummaryThis study mainly employed metabolomics technology to determine changes of intracellular metabolite concentrations related to milk protein synthesis induced by heat stress (HS) in bovine mammary epithelial cells. HS was associated with significant differences in intracellular amino acid metabolism resulting in an increase in the intracellular amino acid concentrations. Moreover, HS promoted amino acid transportation and the activity of the mammalian target of rapamycin (mTOR) signaling pathway, which plays an important role as a central regulator of cell metabolism, growth, proliferation and survival. Greater expression of the alpha-S2-casein gene (CSN1S2) was also observed during HS. Overall, our study indicated that bovine mammary epithelial cells may have the ability to resist HS damage and continue milk protein synthesis partly through enhanced intracellular amino acid absorption and metabolism and by activating the mTOR signaling pathway during HS.Heat stress (HS) is one of the most serious factors to negatively affect the lactation performance of dairy cows. Bovine mammary epithelial cells are important for lactation. It was demonstrated that HS decreases the lactation performance of dairy cows, partly through altering gene expression within bovine mammary epithelial tissue. However, the cellular metabolism mechanisms under HS remains largely unknown. The objective of this study was to determine whether HS induced changes in intracellular metabolites and gene transcription related to amino acid metabolism, amino acid transportation and the mTOR signaling pathway. Immortalized bovine mammary epithelial cell lines (MAC-T cells, n = 5 replicates/treatment) were incubated for 12 h at 37 °C (Control group) and 42 °C (HS group). Relative to the control group, HS led to a greater mRNA expression of heat shock protein genes HSF1, HSPB8, HSPA5, HSP90AB1 and HSPA1A. Compared with the control group, metabolomics using liquid chromatography tandem–mass spectrometry identified 417 differential metabolites with p < 0.05 and a variable importance in projection (VIP) score >1.0 in the HS group. HS resulted in significant changes to the intracellular amino acid metabolism of glutathione, phenylalanine, tyrosine, tryptophan, valine, leucine, isoleucine, arginine, proline, cysteine, methionine, alanine, aspartate and glutamate. HS led to a greater mRNA expression of the amino acid transporter genes SLC43A1, SLC38A9, SLC36A1, and SLC3A2 but a lower mRNA expression of SLC7A5 and SLC38A2. Additionally, HS influenced the expression of genes associated with the mTOR signaling pathway and significantly upregulated the mRNA expression of mTOR, AKT, RHEB, eIF4E and eEF2K but decreased the mRNA expression of TSC1, TSC2 and eEF2 relative to the control group. Compared with the control group, HS also led to greater mRNA expression of the CSN1S2 gene. Overall, our study indicates that bovine mammary epithelial cells may have the ability to resist HS damage and continue milk protein synthesis partly through enhanced intracellular amino acid absorption and metabolism and by activating the mTOR signaling pathway during HS.

Supplemental L-Arginine Improves the Embryonic Intestine Development and Microbial Succession in a Chick Embryo Model.

Early colonization of intestinal microbiota plays an important role in intestinal development. However, the microbial succession at an embryonic stage and its assembly patterns induced by prenatal nutrition are unknown. In the present study, we used a chick embryo model to investigate the effects of in ovo feeding (IOF) of L-arginine (Arg) on the intestinal development and microbial succession of embryos. A total of 216 fertile eggs were randomly distributed into 2 groups including the non-injected control group and IOF of Arg group with 7 mg/egg. The results showed that IOF Arg increased the intestinal index, absolute weight of jejunum, and improved jejunal morphology in terms of villus width and surface area (p < 0.05). The relative mRNA expressions of mTOR and 4E-BP1 were up-regulated and accompanied by higher contents of Mucin-2 in the Arg group (p < 0.05). There was a significant elevation in contents of serum glucose and high-density lipoprotein cholesterol, whereas there was a decreased low-density lipoprotein cholesterol in the Arg group (p < 0.05). Additionally, Proteobacteria and Firmicutes were major intestinal bacteria species at the embryonic stage. However, Arg supplementation targeted to shape assembly patterns of microbial succession and then changed microbial composition (p = 0.05). Meanwhile, several short-chain fatty acids (SCFAs)-producing bacteria, such as Roseburia, Blautia, and Ruminococcus were identified as biomarkers in the Arg group (LDA > 3, p < 0.05). Accordingly, significant elevated concentrations of SCFAs, including lactic acid and formic acid, were observed in the Arg group (p < 0.05), accompanied by the higher concentration of butyric acid (0.05 < p < 0.10). In conclusion, prenatal Arg supplementation improved embryonic intestine development by regulating glucose and lipid homeostasis to supply more energy for chick embryos. The possible mechanism could be the roles of Arg in shaping the microbial assembly pattern and succession of the embryonic intestine, particularly the enrichment of potential probiotics. These findings may contribute to exploring nutritional strategies to establish health-promoting microbiota by manipulating prenatal host-microbe interactions for the healthy development of neonates.

Modulation of Autophagy Signal Pathway in Type 2 Diabetes Mellitus Rat Model Using Phytochemical Extracts

Abstract Background Type 2 DM is a chronic metabolic disease characterized by insulin resistance and beta cell dysfunction. It is an extremely heterogeneous disease in which the insulin resistance and beta cell dysfunction resulted from environmental and genetic factor. Type 2 diabetes is the most prevalent form of diabetes worldwide and account for 90% of cases globally. It represents a major public health threat and considered as a principal cause of morbidity and mortality affecting almost 6% of the world’s population. Patients with type 2 diabetes usually have insulin resistance and relative insulin deficiency and are often associated with a strong genetic predisposition. Objective To retrieve potential miRNA-associated ceRNAs Linked to autophagy, insulin resistance and type 2 DM from Public microarray databases followed by validation of the chosen network in animal model. To evaluate the efficacy of Isorhamentin as a potential therapeutic strategy to modulate the autophagy pathway and IR in type 2 DM. Patients and Methods This study was done at Medical Biochemistry Department, Faculty of Medicine, Ain Shams University during the period from December 2019 – June 2020 and included 50 wistar male rats. 50 Male Wistar rats weighed (140-150g), age nearly two months, were purchased from the Holding Company for Biological Products and Vaccines. They were acclimatized to laboratory environment for at least one week with free access to water and food before induction of diabetes. Experiment for eight weeks duration was planned after a pilot study was performed for determination of the most effective and least toxic STZ dose. Results Our study demonstrated that “Isorhamentin” can be potential treatment in type 2 diabetes mellitus through modulation of Autophagy signaling pathway related genes and ceRNA network. Isorhamentin down-regulates the expression of m-tor mRNA and it up-regulates the expression of miR-1. Conclusion The results of our study Support our previous hypothesis that miR-1 miRNA and mTOR mRNA act as ceRNA network and might play an important role in the pathogenesis of autophagy and insulin resistance and T2DM.And could be potential therapeutic target by isorhamentin to modulate the expression of the deregulated network in type 2 DM.

Acrylamide impairs the developmental potential of germinal vesicle oocytes by inducing mitochondrial dysfunction and autophagy/apoptosis in mice

Background: Acrylamide (ACR), an important endogenous contaminant in carbohydrate-rich foods, has been involved in various negative effects on multiple organ networks, including the reproductive system. Previous studies have reported that ACR affects oocyte quality and fertility.Purpose: This study aimed to explore the toxic effects and regulatory mechanisms of ACR on mouse germinal vesicle (GV) oocytes. Research Design: In this study, adult female mice were exposed to ACR at 10mg/kg/day/body weight through their drinking water continuously for 4weeks. Study Sample and Data Analysis: The mitochondrial function, autophagy/apoptosis, and development potential of GV oocytes were investigated. Results: The results showed that ACR reduced the oocyte diameter, sperm-binding ability, parthenogenetic activation and in vitro fertilization (IVF) rate, and development potential of pre-implantation embryos. We also found that ACR exposure disrupted chromatin configuration, mitochondrial distribution, and membrane potential (Δφm) of oocytes. Actin filament expression was significantly reduced in both the membrane and cytoplasm of mouse oocytes. Moreover, ACR exposure increased LC3-positive signals, early apoptosis rate, aberrant ATG3, ATG5, LC3, Beclin1, and mTOR mRNA expression. Conclusions: These results suggest that ACR exposure can affect the developmental potential of GV oocytes by inducing mitochondrial dysfunction, actin filament assembly, and autophagy/apoptosis.

MiR-149 Alleviates Ox-LDL-Induced Endothelial Cell Injury by Promoting Autophagy through Akt/mTOR Pathway.

Background Atherosclerosis is a chronic process that takes place in the vascular wall and causes various cardiovascular diseases (CVDs). Micro-RNA-149 (miR-149) mediates many physiological and pathological processes, including atherosclerosis. However, it is unclear about the roles of miR-149 in endothelial injury. Here, we explored the protective effect and related mechanism of miR-149 in endothelial cells induced with oxidized low-density lipoprotein (ox-LDL). Methods Human endothelial cell lines (HUVECs) were exposed to ox-LDL to induce endothelial injury. Cell viability was determined by the CCK-8 assay. Autophagy was detected by immunofluorescence. RT-qPCR and western blot were carried out to determine the mRNA and protein expressions of Akt and mTOR. Results The miR-149 level in HUVECs was reduced by ox-LDL (100 μg/mL) incubation in a time-dependent manner. miR-149-mimic transfection markedly protected HUVECs from ox-LDL-induced injury, with increased cell viability and reduced caspase-3 activity. miR-149 mimics enhanced HUVEC autophagy, which was induced initially by ox-LDL. miR-149 mimics also markedly downregulated the expression of Akt, p-Akt, mTOR, and p-mTOR in ox-LDL-treated HUVECs. The miR-149-induced protection against HUVECs injury could be reversed by cotreatment with 3-methyladenine (3-MA, an autophagy inhibitor) or insulin (an activator of Akt/mTOR pathway). Conclusions miR-149 prevents ox-LDL-induced endothelial cell injury by enhancing autophagy via increasing Akt and mTOR expressions.

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.

Purple sweet potato extract maintains intestinal homeostasis and extend lifespan through increasing autophagy in female Drosophila melanogaster.

Purple sweet potatoes extract (PSPE) have been used as a natural food antioxidant with high anthocyanin concentrations. Research investigated the lifespan and the mechanisms of PSPE on female Drosophila melanogaster. Supplementation of PSPE extended the lifespan by 16.3% and had a protective effect on injury by oxidative stress. PSPE treatment enhanced the endogenous antioxidant enzyme activity and reduced malondialdehyde (MDA) content. Furthermore, PSPE significantly up-regulated foxo-related genes, inhibited mTOR mRNA expression, and activated autophagy to maintain intestinal homeostasis. Meanwhile, PSPE improved intestinal barrier dysfunction by 22.86%, decelerated the abnormal proliferation rate of intestinal stem cell (ISCs) by 23.77%, and improved intestinal integrity in geriatric D. melanogaster. In conclusion, PSPE may maintain intestinal homeostasis, and improve the antioxidant and stress resistance capacity through the insulin and rapamycin pathway, thereby extending the lifespan. Therefore, it provides active support to the development and application of PSPE in functional food. PRACTICAL APPLICATIONS: In recent years, with the increase of age, age-related complications have generally increased and seriously affected people's healthy life. Purple sweet potato is a nutrient-rich substance, which not only has a unique color but also contains rich anthocyanins, so it has many potential biological and pharmacological functions. Our results showed that the PSPE had a good effect of maintaining the intestinal homeostasis of the older adult, and provided a favorable theoretical basis for the development of PSPE functional products and scientific academic research.

MiR-424 induces apoptosis in glioblastoma cells and targets AKT1 and RAF1 oncogenes from the ERBB signaling pathway

Glioblastoma is a lethal and incurable cancer. Tumor suppressor miRNAs are promising gene therapy tools for cancer treatment. In silico, we predicted miR-424 as a tumor suppressor. It had several target genes from the epidermal growth factor receptor (ERBB) signaling pathway that are overactive in most glioblastoma cases.We overexpressed miR-424 by lentiviral transduction of U-251 and U-87 glioblastoma cells confirmed with fluorescent microscopy and real-time quantitative PCR (qRT-PCR). Then the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) proliferation assay and scratch wound migration assay were performed to investigate the miR-424 tumor suppressor effect in glioblastoma. miR-424's effect on glioblastoma apoptosis and cell-cycle arrest was verified using Annexin V- phosphatidylethanolamine (PE) and 7-minoactinomycin D (7-AAD) apoptosis assay and cell-cycle assay. miR-424 predicted target genes mRNA and protein level were measured after miR-424 overexpression in comparison to the control group by qRT-PCR and western blotting, respectively. We confirmed miR-424 direct target genes by dual-luciferase reporter assay.miR-424 overexpression significantly suppressed cell proliferation and migration rate in glioblastoma cells based on the MTT and scratch assays. Flow cytometry results confirmed that miR-424 promotes apoptosis and cell-cycle arrest in glioblastoma cells. Predicted target genes of miR-424 from the ERBB pathway were downregulated by miR-424 overexpression. qRT-PCR and western blotting showed that KRAS, RAF1, MAP2K1, EGFR, PDGFRA, AKT1, and mTOR mRNA expression levels and KRAS, RAF1, MAP2K1, EGFR, and AKT1 protein level, respectively, had significantly decreased as a result of miR-424 overexpression in comparison to the control group. Dual-luciferase reporter assay confirmed that miR-424 directly targets RAF1 and AKT1 oncogenes.Overall, miR-424 acts as tumor suppressor miRNA in glioblastoma cells.

Verbascoside inhibits the progression of atherosclerosis in high fat diet induced atherosclerosis rat model.

Atherosclerosis is thickening of arterial wall, which causes several complications. This study evaluated the protective effect of verbascoside against atherosclerosis and the potential molecular mechanism involved. Atherosclerosis was induced by administering a high-fat diet to rats for 3 months and vitamin D3 for 4 days. Verbascoside (2 mg/kg p.o.) was administered for 6 weeks after the end of high-fat diet administration. The serum levels of inflammatory mediators and the lipid profile were determined, and atherosclerotic lesions were observed via Sudan IV staining. Moreover, immunohistochemical analysis, Western blot assay and qRT-PCR were performed to determine the effect of verbascoside on the AMPK/mTOR pathway in atherosclerosis. The results reveal that verbascoside ameliorated the altered serum levels of inflammatory mediators and the lipid profile as well as the organ coefficients in the atherosclerotic rats. A significant decrease in atherosclerotic lesions was detected in the verbascoside-treated group than the atherosclerosis group. AMPK and mTOR mRNA expression decreased in aortic tissues of the verbascoside-treated group compared to the atherosclerosis group. These data suggest that verbascoside ameliorates atherosclerosis by decreasing hyperlipidaemia in atherosclerosis induced by a high-fat diet.

Effect of electroacupuncture on the expression of autophagy related protein in lung tissue of rats with chronic obstructive pulmonary disease

To observe the effect of electroacupuncture (EA) at "Zusanli" (ST36) and "Feishu" (BL13) on the expression of autophagy related proteins in the lung tissue of rats with chronic obstructive pulmonary disease (COPD), so as to explore the mechanism of EA underlying improvement of COPD. Thirty male SD rats were randomly divided into normal, model and EA groups (n=10 in each group). The COPD model was established by intratracheal infusion of Lipopolysaccharide (LPS, 1 mg/kg) and exposure in cigarette smoke. EA was applied to bilateral ST36 and BL13 for 30 min, once every other day for 2 weeks. The pulmonary function (forced vital capacity ［FVC］, forced expiratory volume in 0.1 s and 0.3 s ［FEV0.1, FEV0.3］, FEV0.1/FVC and FEV0.3/FVC) was detected by animal pulmonary function analysis system. Histopathological changes of the airway and lung were displayed by H.E. staining. Autophagosomes in the airway and lung tissues were observed by electron microscope. The expression of AMP activated protein kinase (AMPK), mammalian target of rapamycin (mTOR), Unc-51 like autophagy activating kinase 1(ULK1), autophagy related protein ATG6(Beclin1)mRNAs in lung tissue were examined by quantitative real-time PCR. The expression of AMPK, mTOR, ULK1, Beclin1 and microtubule-associated protein 1 light chain 3 (LC3)proteins in lung tissue were examined by Western blot. The contents of tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) in the broncho alveolar lavage fluid (BALF) were assayed by ELISA. Following modeling, the FVC, FEV0.1, FEV0.3, FEV0.1/FVC and FEV0.3/FVC levels were significantly decreased (P<0.01), the infiltration of inflammatory cells and the increase of autophagosomes were obvious in airway and lung tissue, the mRNA and protein expression of AMPK, ULK1, Beclin1 and the ratio of LC3Ⅱ/LC3Ⅰ were increased (P<0.01), while the mRNA and protein expression of mTOR were decreased (P<0.01), the contents of TNF-α and IL-6 in the BALF were increased in the model group compared with the normal group (P<0.01). After EA intervention, all the indexes mentioned above were completely reversed in the EA group relevant to the model group (P<0.01, P<0.05). EA at ST36 and BL13 can improve the lung function of COPD rats, which may be related to its effects in inhibiting the autophagy level and reducing the inflammation response in the lung.

LONG-TERM CALORIC RESTRICTION IMPROVES MYOCARDIAL SIRT1/AMPK/PGC-1A PATHWAY IN C57BL/6J MICE

Objective: Caloric restriction (CR) is capable of improving heart function, there is as yet no consensus on the mechanism of effect of CR. Silent mating-type information regulation 1 (SIRT1), adenosine monophosphate-activated protein kinase (AMPK), and mTOR are key players in metabolic stress management. We aimed to explore the effect of CR on myocardial SIRT1/AMPK/mTOR pathway in mice. Design and method: Thirty 6-week-old male C57BL/6J mice were randomly divided into three groups: normal control group (NC group, n = 12), high-energy group (HE group, n = 12) and CR group (n = 12) according to different diet. After 11 months, western blot was used to examine proteins such as p-AMPK, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1a), SIRT1, and p-mTOR, whereas real-time PCR was used to examine the expression of AMPK, PGC-1a, and SIRT1 transcripts. Results: Compared with NC group, the CR group displayed increased expression of myocardial p-AMPK, SIRT1 protein and mRNA, and PGC-1a mRNA. However, no difference was observed in the expression of p-mTOR protein and mTOR mRNA in the myocardium among the three groups. Conclusions: CR improves SIRT1/AMPK/PGC-1a pathway in mice myocardium with no effect on the mTOR pathway.