ATG5 Expression Research Articles

The Effect of Mesenchymal Stem Cells on Dry Eye in Sjogren Syndrome Mouse Model.

Sjögren's syndrome (SS) is a systemic autoimmune disease delineated by chronic lymphocytic infiltrates into the lacrimal or salivary glands, leading to severe dry eye and dry mouth. Mesenchymal stem cells have been shown to be effective in treating numerous autoimmune diseases. This study aimed to illustrate the effects of mesenchymal stem cells on the attenuation of dry eyes (DE) through the inhibition of autophagy markers in a SS mouse model. NOD/ShiLtJ female mice with developed DE were treated with either subconjunctival or lacrimal gland injections of hMSCs (Catholic MASTER Cells). After maintenance for 14 days, clinical DE markers such as tear secretion and corneal staining were observed, as well as goblet cell counts in the conjunctiva, infiltration of inflammatory foci, B and T cells, and autophagy markers in the lacrimal glands. Proinflammatory cytokine expressions of the cornea and conjunctiva, as well as the lacrimal glands, were examined. Clinical markers, such as tear secretion and corneal stain scores, goblet cell counts in the conjunctiva, and foci infiltrations in the lacrimal glands were attenuated in mice treated with subconjunctival or lacrimal gland injections of hMSCs compared to the PBS-treated control group. B cell marker B220 decreased in the lacrimal glands of hMSCs-treated mice, as well as reduced proinflammatory cytokine expressions in the lacrimal glands and cornea. Notably, expression of autophagy markers ATG5 and LC3B-II, as well as HIF-1α and mTOR which play roles in the pathways of autophagy modulation, were shown to be attenuated in the lacrimal glands of hMSCs-treated mice compared to the PBS-treated control mice. Treatment with hMSCs by lacrimal gland or subconjunctival injection demonstrated the alleviation of DE through the repression of autophagy markers, suggesting the therapeutic potentials of hMSCs in a SS mouse model.

Betulinic acid may modulate autophagy in renal cell carcinoma cells.

Betulinic acid is pentacyclic triterpenoid known to exert antitumor effects by modulating many cellular pathways in various human malignancies. However, its modulatory role in autophagy in renal cell carcinoma remains unclear. Here, we observed how betulinic acid affects autophagy in renal cell carcinoma cells. After treating cells with betulinic acid, we determined the gene expression and protein levels of Beclin-1 and ATG-5 by qPCR and ELISA assay to observe its effects on autophagy. The qPCR results demonstrated that Beclin-1 expression level was low in untreated metastatic renal adenocarcinoma ACHN cells and increased in response to 25 µM and 50 µM betulinic acid treatment. ATG-5 expression level was decreased in primary clear cell renal cell carcinoma CAKI-2 cells treated with 50 µM betulinic acid. In the ELISA assay results, we observed that betulinic acid caused a decrease in Beclin-1 protein level at 25 µM concentration and in ATG-5 protein level at 50 µM concentration in CAKI-2 cells. In our preliminarily study, it was concluded that the role of autophagy may differ in renal cell carcinoma cells depending on their origin and that the effects of betulinic acid on autophagy in these cells may vary accordingly (Fig. 4, Ref. 40). Text in PDF www.elis.sk Keywords: betulinic acid, autophagy, kidney, cancer, cell.

Regulation of autophagy in chick myotube cultures: Effect of uncoupling mitochondrial oxidative phosphorylation.

Abstracts: Skeletal muscles have a high demand for ATP, which is met largely through mitochondria oxidative phosphorylation. Autophagy is essential for the maintenance of skeletal muscle mass under catabolic conditions. This study investigated the effect of uncoupling mitochondrial oxidative phosphorylation on autophagy in chicken skeletal muscle. Chick myotubes were incubated with the mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP) at 25 μM for 3h. CCCP prevented the phosphorylation of p70 ribosomal S6 kinase 1 (Thr389), S6 ribosomal protein (Ser240/244), and eukaryotic translation initiation factor 4E-binding protein 1 (Thr37/46), which are the measures of the mechanistic target of rapamycin complex 1 (mTORC1) activity. CCCP significantly increased cytoplasmic and mitochondrial LC3-II content, which act as indices of index for autophagosome formation and mitophagy, respectively, but did not influence the expression of autophagy-related genes LC3B, GABARAPL1, and ATG12. Finally, surface sensing of translation method revealed that protein synthesis, a highly energy consuming process, was significantly decreased upon CCCP treatment. These results indicate that the uncoupling of mitochondrial oxidative phosphorylation stimulates autophagy and inhibits protein synthesis through mTORC1 signaling in chick myotube cultures.

Hyperactive neuronal autophagy depletes BDNF and impairs adult hippocampal neurogenesis in a corticosterone-induced mouse model of depression.

Background: Depression is a mental disorder that poses a serious threat to human health. Adult hippocampal neurogenesis (AHN) is closely associated with the efficacy of antidepressants. Chronic treatment with corticosterone (CORT), a well-validated pharmacological stressor, induces depressive-like behaviors and suppresses AHN in experimental animals. However, the possible mechanisms of chronic CORT action remain elusive. Methods: A chronic CORT treatment (0.1 mg/mL, drinking water for 4 weeks) was applied to prepare a mouse model of depression. Immunofluorescence was performed to analyze the hippocampal neurogenesis lineage, and immunoblotting, immunofluorescence, electron microscopy, and adeno-associated virus (AAV) expressing a pH-sensitive tandemly tagged light chain 3 (LC3) protein were used to analyze neuronal autophagy. AAV-hSyn-miR30-shRNA was used to knock down autophagy-related gene 5 (Atg5) expression in the neurons. Results: Chronic CORT induces depressive-like behaviors and decreases the expression of neuronal brain-derived neurotrophic factor (BDNF) in the dentate gyrus (DG) of the hippocampus in mice. Moreover, it markedly diminishes the proliferation of neural stem cells (NSCs), neural progenitor cells, and neuroblasts and impairs the survival and migration of newborn immature and mature neurons in the DG, which may be attributed to changes in the cell cycle kinetics and induction of NSCs apoptosis. Furthermore, chronic CORT induces hyperactive neuronal autophagy in the DG, possibly by increasing the expression of ATG5 and causing excess lysosomal degradation of BDNF in neurons. Notably, inhibiting hyperactive neuronal autophagy in the DG of mice by knocking down Atg5 in neurons using RNA interference reverses the decrease of neuronal BDNF expression, rescues AHN, and exerts antidepressant effects. Conclusion: Our findings reveal a neuronal autophagy-dependent mechanism that links chronic CORT to reduced neuronal BDNF levels, AHN suppression and depressive-like behavior in mice. In addition, our results provide insights for treating depression by targeting neuronal autophagy in the DG of the hippocampus.

Dihydromyricetin supplementation during in vitro culture improves porcine oocyte developmental competence by regulating oxidative stress.

Dihydromyricetin (DHM), a dihydroflavonoid compound, exhibits a variety of biological activities, including antitumor activity. However, the effects of DHM on mammalian reproductive processes, especially during early embryonic development, remain unclear. In this study, we added DHM to porcine zygotic medium to explore the influence and underlying mechanisms of DHM on the developmental competence of parthenogenetically activated porcine embryos. Supplementation with 5 μM DHM during in vitro culture (IVC) significantly improved blastocyst formation rate and increased the total number of cells in porcine embryos. Further, DHM supplementation also improved glutathione levels and mitochondrial membrane potential; reduced natural reactive oxygen species levels in blastomeres and apoptosis rate; upregulated Nanog, Oct4, SOD1, SOD2, Sirt1, and Bcl2 expression; and downregulated Beclin1, ATG12, and Bax expression. Collectively, DHM supplementation regulated oxidative stress during IVC and could act as a potential antioxidant during in vitro porcine oocytes maturation.

Methyltransferase-like 3 suppresses phenotypic switching of vascular smooth muscle cells by activating autophagosome formation.

Prevention of neointima formation is the key to improving long-term outcomes after stenting or coronary artery bypass grafting. RNA N6 -methyladenosine (m6 A) methylation has been reported to be involved in the development of various cardiovascular diseases, but whether it has a regulatory effect on neointima formation is unknown. Herein, we revealed that methyltransferase-like 3 (METTL3), the major methyltransferase of m6 A methylation, was downregulated during vascular smooth muscle cell (VSMC) proliferation and neointima formation. Knockdown of METTL3 facilitated, while overexpression of METTL3 suppressed the proliferation of human aortic smooth muscle cells (HASMCs) by arresting HASMCs at G2/M checkpoint and the phosphorylation of CDC2 (p-CDC2) was inactivated by METTL3. On the other hand, the migration and synthetic phenotype of HASMCs were enhanced by METTL3 knockdown, but inhibited by METTL3 overexpression. The protein levels of matrix metalloproteinase 2 (MMP2), MMP7 and MMP9 were reduced, while the expression level of tissue inhibitor of metalloproteinase 3 was increased in HASMCs with METTL3 overexpression. Moreover, METTL3 promoted the autophagosome formation by upregulating the expression of ATG5 (autophagy-related 5) and ATG7. Knockdown of either ATG5 or ATG7 largely reversed the regulatory effects of METTL3 overexpression on phenotypic switching of HASMCs, as evidenced by increased proliferation and migration, and predisposed to synthetic phenotype. These results indicate that METTL3 inhibits the phenotypic switching of VSMCs by positively regulating ATG5-mediated and ATG7-mediated autophagosome formation. Thus, enhancing the level of RNA m6 A or the formation of autophagosomes is the promising strategy to delay neointima formation.

Study of the Regulatory Mechanism of miR-26a-5p in Allergic Asthma.

Allergic asthma is a growing burden on national public health services due to its high prevalence. The aim of this experiment was to investigate whether miR-26a-5p affects cellular fibrosis and thus airway remodeling in asthmatic mice through the regulation of target genes. Screening for differentially expressed miRNAs in asthma model mice was carried out by constructing a mouse model of allergic asthma. qRT-PCR was performed to determine candidate miRNAs in each group of bronchial tissues. Western blot detection of the expression levels of predicted candidate target genes in each group of bronchial tissues was conducted. A dual luciferase assay was performed to validate the binding of miR-26a-5p to target genes. Fibronectin, a marker of cellular fibrosis, was detected via flow cytometry. CCK8 and BrdU staining were used to detect the proliferation ability of each group of cells. miR-26a-5p is able to target and bind to ABL2 3'-UTR, MMP16 3'-UTR and PDE7A 3'-UTR sequences. After interference with miR-26a-5p, improved bronchial histopathology and reduced peribronchial collagen deposition were found. Compared with the model group, interference with miR-26a-5p reduced lung fibrosis, decreased fibroblasts and increased apoptosis in mouse bronchial tissues; overexpression of miR-26a-5p decreased apoptosis in mouse bronchial tissues. Compared with the model group, the serum levels of IL-4, IL-5, IL-13 and I IFN-γ were decreased in the miR-26a-5p inhibitor group and increased in the miR-26a-5p mimic group. The immunohistochemical results showed that the expression of ABL2, MMP16 and PDE7A was significantly reduced after intervention with miR-26a-5p. Compared with the model group, the apoptosis rate of cells in the miR-26a-5p inhibitor group of the allergic asthma model was upregulated, the levels of IL-4, IL-5, IL-13, IFN-γ and ROS were decreased, the expression of the miRNA and proteins of ABL2, MMP16 and PDE7A was decreased, the expression of LC3A and P62 was significantly increased and the expression of LC3B, Beclin1, Atg5 and fibrosis markers collagen I and α-SMA was decreased. miR-26a-5p affects cellular fibrosis and thus airway remodeling in asthmatic mice by regulating target genes.

Non-cytopathic bovine viral diarrhoea virus 2 induces autophagy to enhance its replication.

Bovine viral diarrhoea virus (BVDV) is an important viral pathogen that has an economic impact on the livestock industry worldwide. Autophagy is one of the earliest cell-autonomous defence mechanisms against microbial invasion, and many types of viruses can induce autophagy by infecting host cells. The aim of this study was to identify the role of autophagy in the pathogenesis of non-cytopathic (ncp) BVDV2 infection. Madin-Darby bovine kidney (MDBK) cells were treated with ncp BVDV2, rapamycin, or 3-methyladenine (MA) and ncp BVDV2 and then incubated at 37°C for 24h. Cells were harvested, and the effects of autophagy were determined by transmission electron microscopy (TEM), confocal laser microscopy, western blotting and qRT-PCR. Apoptotic analysis was also performed using western blotting and flow cytometry. In ncp BVDV2-infected MDBK cells, more autophagosomes were observed by TEM, and the number of microtubule-associated protein 1 light chain 3B (LC3B) with green fluorescent protein puncta was also increased. The ncp BVDV2-infected cells showed significantly enhanced conversion of LC3-I to LC3-II, as well as upregulation of autophagy-related proteins, including ATG5 and Beclin 1, and substantial degradation of p62/SQSTM1. These results are similar to those induced by rapamycin, an autophagy inducer. E2 protein expression, which is associated with viral replication, increased over time in ncp BVDV2-infected cells. Inhibition of autophagy by 3-MA in ncp BVDV2-infected MDBK cells downregulated the expressions of LC3-II, ATG5 and Beclin 1 and prevented the degradation of p62/SQSTM1. Moreover, the expressions of phosphorylated Akt and procaspase-3 were significantly increased in ncp BVDV2-infected cells. In addition, the mRNA level of protein kinase R (PKR) was significantly reduced in ncp BVDV2-infected cells. Our results demonstrate that ncp BVDV2 infection induced autophagy in MDBK cells via anti-apoptosis and PKR suppression. Therefore, autophagy may play a role in establishing persistent infection caused by ncp BVDV.

Inducing cathepsin L expression/production, lysosomal activation, and autophagy of human dental pulp cells by dentin bonding agents, camphorquinone and BisGMA and the related mechanisms

Camphorquinone (CQ) and resin monomers are included in dentin bonding agents (DBAs) and composite resin to restore tooth defects due to abrasion, crown fracture, or dental caries. DBAs, CQ, and bisphenol A-glycidyl methacrylate (BisGMA) applications influence the biological activities of the dental pulp. The current investigation aimed to delineate the effect of DBAs, CQ, and BisGMA on cathepsin L production/expression, lysosomal activity, and autophagy induction in human dental pulp cells (HDPCs). HDPCs were exposed to DBAs, CQ, or BisGMA with/without inhibitors for 24 h. Enzyme-linked immunosorbent assay was employed to determine the cathepsin L level in culture medium. The cell layer was utilized to measure cell viability by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl -tetrazolium bromide (MTT) assay. Real-time PCR was used to evaluate the mRNA expression. Western blotting or immunofluorescent staining was used to study protein expression. Lysosomal density was evaluated by lysotracker red staining. We found that DBAs, CQ, and BisGMA stimulated cathepsin L mRNA, protein expression, and production in HDPCs. In addition, CQ and BisGMA induced lysosomal activity, Beclin1, ATG12, LC3B, Bax, and p53 expression in HDPCs, indicating the stimulation of autophagy. Glutathione (GSH) prevented CQ- and BisGMA-induced cytotoxicity. Moreover, E64d, cathepsin L inhibitor (two cathepsin inhibitors), and Pifithrin-α (a p53 inhibitor) showed little preventive effect toward CQ- and BisGMA-induced cytotoxicity. Autophagy inhibitors (NH4Cl, Lys05) mildly enhanced the CQ- and BisGMA-induced cytotoxicity. These results indicate that DBAs stimulated cathepsin L, possibly due to their content of CQ and BisGMA that may induce cathepsin L in HDPCs. CQ and BisGMA stimulated lysosomal activity, autophagy, and apoptosis, possibly via induction of Beclin 1, ATG12, LC-3B, Bax, and p53 expression. In addition, CQ and BisGMA cytotoxicity was related to redox change and autophagy. These events are important role in pulpal changes after the restoration of tooth decay using CQ- and BisGMA-containing DBAs and resin composite.

Trehalose promotes functional recovery of keratinocytes under oxidative stress and wound healing via ATG5/ATG7

Wounds are in a stressed state, which precludes healing. Trehalose is a stress metabolite that protects cells under stress. Here, we explored whether trehalose reduces stress-induced wound tissue damage. A stress model was prepared by exposing human keratinocytes to hydrogen peroxide (H2O2), followed by trehalose treatment. Trehalose effects on expression of the autophagy-related proteins ATG5 and ATG7 and cell proliferation and migration were evaluated. For in vivo verification, a wound model was established in Sprague–Dawley rats, to measure the effects of trehalose wound-healing rate and reactive oxygen species (ROS) content. Histological changes during wound healing and trehalose's effects on ATG5 and ATG7 expression, necrosis, and apoptosis were examined·H2O2 stress increased ATG5 and ATG7 expression in vitro, but this was insufficient to prevent stress-induced damage. Trehalose further increased ATG5/ATG7 levels, which restored proliferation and increased migration by depolymerizing the cytoskeleton. However, trehalose did not exert these effects after ATG5 and ATG7 knockout. In vivo, the ROS content was higher in the wound tissue than in normal skin. Trehalose increased ATG5/ATG7 expression in wound tissue keratinocytes, reduced necrosis, depolymerized the cytoskeleton, and promoted cell migration, thereby promoting wound healing.

Quercetin Ameliorates Lipopolysaccharide-Induced Duodenal Inflammation through Modulating Autophagy, Programmed Cell Death and Intestinal Mucosal Barrier Function in Chicken Embryos.

Diarrhea has been a global health problem for centuries, and the treatment has become increasingly difficult duo to the antibiotics overuse and resistance. Quercetin is a common flavonoid of extracts of vegetables, fruits, and traditional Chinese herbs, however, the mechanism of quercetin alleviating LPS-induced duodenal inflammation remains elusive. Specific pathogen-free chicken embryos (n = 120) were allocated to groups including control, PBS with or without alcohol, LPS (125 ng/egg) with or without quercetin (10, 20, or 40 nmol/egg, respectively), and quercetin groups (10, 20, or 40 nmol/egg). Fifteen day-old embryonated eggs were inoculated with abovementioned solutions via the allantoic cavity. At embryonic day 19, the duodena of the embryos were collected for histopathological examination, RNA extraction and real-time polymerase chain reaction, immunohistochemical investigations, and Western blotting. The results demonstrated quercetin enhanced the inflammatory cell infiltration in the Peyer's patch of the intestinal mucosa after LPS induction. The LPS-induced expressions of these inflammation-related factors (TLR4, IL-1β, MMP3, MMP9, NFKB1, IFNγ, IL-8, IL-6) were completely blocked by quercetin. Quercetin also decreased the protein expression of TLR4, IL-1β, MMP3, and MMP9 after LPS induction. Quercetin could down-regulate autophagy gene expression (ATG5, LC3-1, LC3-2, and LKB1), and decreased the protein expression of ATG5, and LC3-1/LC3-2 after LPS induction. Quercetin treatment prevented LPS-induced increases of the gene expressions of programmed cell death factors (TNFα, Fas, CASP1, CASP3, CASP12, Drp1, and RIPK1); meanwhile, quercetin decreased the protein expression of CASP1 and CASP3 after LPS challenge. LPS reduced the gene expression of mucin 2, but upregulated the mRNA and protein expression of claudin 1, occludin, and ZO-1, and this was balanced by quercetin. This evidence suggests that quercetin can alleviate duodenal inflammation induced by LPS through modulating autophagy, programmed cell death, intestinal barrier function.

Impaired Autophagy Causes Severe Corneal Neovascularization.

To investigate the role of macrophage autophagy in the process of corneal neovascularization (CNV). In vivo, mice CNV was induced by alkali injury and compared with rapamycin-treated alkaline burn mice. Western blot was used to determine the autophagic status of the macrophages. We quantified the levels of macrophage polarization markers (CD86, INOS, CD163, CD206) by RT-qPCR and measured inflammatory factors through ELISA (IL-6 and TNF-α) in the early phase after injury. In vitro, the human umbilical vein endothelial cells (HUVECs) were co-cultured with macrophage-conditioned medium (MCM) induced by the THP-1 cell line to simulate the neovascular microenvironment. The vascularization capacity of HUVECs was examined using the CCK-8 assay kit, tube formation assay, and scratch wound-healing assay. In vivo, the mRNA expression of Beclin-1 and ATG5 was increased, together with the upregulation of M1 macrophage markers (CD86 and INOS) in corneas after early alkali injury. The area of CNV is effectively relieved in the rapamycin-treated mice. In vitro, upregulation of autophagy level by pretreatment with 3-methyladenine (3-MA) could increase the mRNA expression of the M1 markers. Macrophage-conditioned medium with impaired autophagy contains more IL-6 and TNF-α compared to the M1 macrophage-conditioned medium, promoting HUVEC proliferation, migration, and tube formation capacity. Enhancing the autophagy level with rapamycin (RAPA) could reverse this phenomenon. Impaired autophagy promoted macrophage polarization toward M1 type and increased the expression of IL-6 and TNF-α, which led to severe CNV. Using the autophagy activator (RAPA) could effectively alleviate CNV by promoting autophagy.

Cuproptosis key gene FDX1 is a prognostic biomarker and associated with immune infiltration in glioma.

Recent studies have found that the protein encoded by the FDX1 gene is involved in mediating Cuproptosis as a regulator of protein lipoylation and related to immune response process of tumors. However, the specific biological function of FDX1 in glioma is currently unclear. To explore the potential function of FDX1, this study explored the correlation between the expression of FDX1 in cancers and survival prognosis by analyzing the public databases of GEPIA and Cbioportal. Immune infiltration was analyzed by the TIMER2.0 database in tumors. The possible biological processes and functions of FDX1-related in glioma were annotated through gene enrichment. Relationship between Cuproptosis and autophagy was explored through gene co-expression studies. Summary and conclusions of this study: (1) FDX1 is highly expressed in gliomas and associated with poor prognosis in low-grade gliomas (LGG). (2) Gene annotation indicates that FDX1 is mainly involved in the tumor protein lipoylation and cell death. (3) FDX1 expression is positively correlated with the infiltration of immune cells. (4) LIPT2 and NNAT, two other genes involved in lipoylation, may be unidentified marker gene for Cuproptosis. And the Cuproptosis genes related to FDX1 were positively correlated with the expression of autophagy marker genes Atg5, Atg12, and BECN-1. This evidence suggests that there may be some interaction between FDX1 mediated Cuproptosis and autophagy. In summary, FDX1 may serve as a potential immunotherapy target and prognostic marker for Glioma.

Effects of dichloromethane extraction from Piper nigrum L. and P. longum L. on the expression of autophagy-related proteins in ischemic stroke

Piper nigrum L. and P. longum L. are widely used in various medicinal formulations. The dichloromethane fraction of Piper nigrum L. and P. longum L. (DF) can prevent cerebral ischemic injury although the underlying mechanisms are obscure. The aim of this study was to evaluate the potential neuroprotective effects of DF on a rat model of permanent middle cerebral artery occlusion (pMCAO) and assess the molecular mechanisms. Animals were administered with DF (50, 100, and 150 mg/kg) or nimodipine (12 mg/kg) 6 h after pMCAO for 14 consecutive days via intragastric gavage. In the vitro this study identified that DF reduced neurological severity scores and improved survival rate. Results showed that DF markedly inhibited the percentage of apoptotic cells as well as neuronal autophagy and mitigated the overall neuronal and vascular damage in the ischemic region. Western blot testing showed that at the molecular level, DF significantly suppressed ischemia-induced activated expression of LC3, Beclin1, Atg12, and Atg5. Overall, our study indicated that DF attenuated neuronal autophagy by suppressing the expression of autophagy-related proteins to generate neuroprotection effect for ischemic stroke.

Sulforaphane Ameliorates Hepatic Lipid Metabolism via Modulating Lipophagy In Vivo and In Vitro.

Although sulforaphane (SFN) is reported to ameliorate the excessive accumulation of lipid droplets (LDs) in hepatocytes, its underlying mechanism remains unclear. This paper aims to investigate how SFN induces hepatic LD degradation via activating macroautophagy. High-fat diet and free fatty acids (FFAs) were used to induce excessive LD formation in hepatocytes in vivo and in vitro, respectively. SFN-induced macroautophagy was shown by the increased LC3 protein expression both (1.32 ± 0.18) in vivo and (2.43 ± 0.22) in vitro. The mRNA levels of Lc3 (1.99 ± 0.16), Atg4 (2.12 ± 0.23), Ulk1 (1.19 ± 0.12), Atg7 (1.25 ± 0.11), and Atg5 (0.81 ± 0.1) genes were elevated by SFN. SFN individually enhanced the localization of LC3 (0.41 ± 0.15), LAMP1 (0.66 ± 0.14), ATG7 (0.26 ± 0.08), and ATG5 (0.38 ± 0.09) with LDs, indicating the occurrence of lipophagy. In the components of LDs isolated from SFN treatment, the expressions of LC3, ATG7, and ATG5 protein were largely increased both in vivo and in vitro. LDs were visualized in autophagosomes which confirmed that the lipophagy was triggered by SFN. Moreover, SFN treatment improved the profile of FFAs which was characterized by increasing the FFAs in liver (total FFA: 261.51 ± 39.58 μM/g) and serum (total FFA: 967.59 ± 239.18 nM/mL). After silencing the nrf2 gene, ATG7 and ATG5 protein expressions were decreased and attenuated this induction by SFN. Nrf2 gene silencing inversely increased TG contents. In summary, SFN enhanced the LD degradation via stimulating lipophagy in a Nrf2-dependent manner.

Autophagy-related genes in Egyptian patients with Behçet's disease

BackgroundBehçet's disease (BD) is a chronic, multi-systemic, recurrent condition that affects the vascular, ocular, mucocutaneous, and central nervous systems. The diagnosis of this disease depends on its clinical features, which are similar to those observed in several diseases, such as Parkinson’s disease, pemphigus vulgaris, systemic lupus erythematosus, Crohn ҆s disease, and Sjӧgren’s syndrome. Lysosome-mediated autophagy is a catabolic, cytoprotective mechanism that maintains cell homeostasis by degrading undesired long-lived proteins and recycling nutrients. The aim of this study was to evaluate the correlations between some autophagy-related genes (ATG5, ATG7, ATG12, LC3b, mTOR) and the pathogenesis and immunopathology of BD. The expression levels of the genes were evaluated by quantitative polymerase chain reaction (qPCR) in 101 individuals that are classified into two groups. Group 1: (n = 71) BD patients, Group 2: (n = 30) healthy controls.ResultsPatients with BD had lower mRNA expression levels of ATG5 and mTOR and higher levels of LC3b mRNA than the controls. No significant differences in the levels of both ATG7 and ATG12 were observed between the two groups. According to the area under the curve analysis, LC3b was considered the best candidate biomarker among the selected markers for the diagnosis of BD. The mRNA expression of ATG5 was significantly correlated with patient age and the presence of oral ulcers. The mRNA expression of ATG7 was significantly associated with age and the presence of erythema nodosum and vascular lesions, whereas that of LC3b was significantly correlated with the presence of pustules.ConclusionThese findings indicated that elevated levels of LC3b were strongly associated with BD. Likewise, the levels of ATG5 and ATG7 were associated with the complications and outcomes of this disease. Additional assessments of the mRNA expression levels of these autophagy-related genes might prove beneficial in diagnosing this autoimmune disorder.

Co-exposure to molybdenum and cadmium triggers pyroptosis and autophagy by PI3K/AKT axis in duck spleens.

Excessive amounts of molybdenum (Mo) and cadmium (Cd) are toxicant, but their combined immunotoxicity are not clearly understood. To estimate united impacts of Mo and Cd on pyroptosis and autophagy by PI3K/AKT axis in duck spleens, Mo or/and Cd subchronic toxicity models of ducks were established by feeding diets with different dosages of Mo or/and Cd. Data show that Mo or/and Cd cause oxidative stress by increasing MDA concentration, and decreasing T-AOC, CAT, GSH-Px and T-SOD activities, restrain PI3K/AKT axis by decreasing PI3K, AKT, p-AKT expression levels, which evokes pyroptosis and autophagy by elevating IL-1β, IL-18 concentrations and NLRP3, Caspase-1, ASC, GSDME, GSDMA, NEK7, IL-1β, IL-18 expression levels, promoting autophagosomes, LC3 puncta, Atg5, LC3A, LC3B, LC3II/LC3I and Beclin-1 expression levels, and reducing expression levels of P62 and Dynein. Furthermore, the variations of abovementioned indexes are most pronounced in co-treated group. Overall, results reveal that Mo or/and Cd may evoke pyroptosis and autophagy by PI3K/AKT axis in duck spleens. The association of Mo and Cd exacerbates the changes.

Swainsonine-induced vacuolar degeneration is regulated by mTOR-mediated autophagy in HT22 cells

The indolizidine alkaloid, swainsonine (SW), is the main toxic component of locoweed, which can cause locoism in animals with characteristic neurological dysfunction. Pathological manifestations at cellular level include extensive vacuolar degeneration. Studies have shown that SW can induces autophagy, but the role and mechanism of autophagy in SW-induced vacuolar degeneration is unclear. In this study, we analyzed the role of autophagy in SW-induced cell injury in mouse hippocampal neurons cell line (HT22) using western blotting, qRT-PCR, transmission electron microscopy and immunofluorescence microscopy. The results showed that the expressions of LC3-II, ATG5, Beclin1 and p62 proteins and their mRNAs in HT22 cells were induced by SW treatment. The SW treatment increased the number of autophagosomes with enhanced fluorescence intensity of monodansylcadaverine (MDC) and LC3-II in a time-dose dependent manner. The results of lysosome staining showed that SW could increase the number of lysosomes, increase the intraluminal pH. Transmission electron microscopy results indicate that SW induced autophagosomes, and Baf A1 could effectively alleviate SW-induced vacuolar degeneration. At the molecular level, SW treatment inhibited the expression of p-PI3K, p-AKT, p-ERK, p-AMPK, p-mTOR, p-p70S6K and p-4EBP1 and promoted the expression of p53. Our results collectively suggest, PI3K/AKT/mTOR, ERK/mTOR and p53/mTOR signaling pathways are involved in the regulation of SW-induced autophagy in HT22 cells, while the AMPK/mTOR signaling pathway is not involved in this regulation. Inhibition of autophagic degradation can effectively alleviate SW-induced vacuolar degeneration.

Wenhua Juanbi Recipe Attenuates Rheumatoid Arthritis via Inhibiting miRNA-146a-Mediated Autophagy.

Wenhua Juanbi Recipe (WJR) is widely used for the treatment of rheumatoid arthritis (RA) in China. However, its mechanism of action remains unclear. This study was designed to investigate the potential therapeutic effects of WJR on the proliferation and apoptosis of synovial fibroblasts in RA and its efficacy in inhibiting miRNA-146a-mediated cellular autophagy. A collagen-induced arthritis (CIA) Wistar rat model was established. The model rats were administered WJR or methotrexate (MTX) to assess the therapeutic effect of the drugs. The chemical components of WJR were analyzed using UPLC-Q/TOF-MS. Histological changes; miRNA-146a, ATG5, ATG7, ATG12, Beclin1, LC3II, Bax, and Bcl2 expression; synovial apoptosis; and cellular proliferation were assessed. Primary synovial fibroblasts (FLS) were cultured in vitro using tissue block and transfected with miRNA-146a; an autophagy inducer was added to FLS, inhibiting the PI3K/AKT/mTOR pathway. FLS were cocultured with WJR-containing serum to observe the effects of miRNA-146a-mediated autophagy via the PI3K/AKT/mTOR pathway on CIA-affected rats. Forty and thirty-one compounds were identified in WJR in the positive and negative ion modes, respectively. WJR significantly reduced toe swelling, arthritis scores, and expression of miRNA-146a and autophagy genes (ATG5, ATG7, ATG12, Beclin1, LC32, and Bcl2). Moreover, Bax expression, apoptosis, and attenuated proliferation were observed in rats. WJR could, therefore, regulate autophagy by influencing the miRNA-146a-mediated PI3K/AKT/mTOR pathway, which induces apoptosis and proliferation of FLS. WJR can inhibit autophagy, apoptosis, and proliferation in a CIA rat model by inhibiting the miRNA-146a-mediated PI3K/AKT/mTOR pathway.

LncRNA DDX11-AS1 Promotes Chemoresistance through LIN28A-Mediated ATG12 mRNA Stabilization in Breast Cancer

Introduction: During breast cancer chemotherapy, the chemoresistance that frequently accompanies the treatment has become a big challenge. Long noncoding RNAs (LncRNAs) have been related to the development of chemoresistance in multiple cancer types. LncRNA DDX11-AS1 has shown a carcinogenic role in lung and colorectal cancer and was reported to enhance oxaliplatin resistance in gastric cancer and Taxol insensitivity in esophageal cancer. But its role in breast cancer chemotherapy drug resistance remains unknown. This study aimed to investigate the function and mechanism of lncRNA DDX11-AS1 in breast cancer chemoresistance. Methods: The relationship between DDX11-AS1 and adriamycin (ADR) resistance was confirmed by qPCR, cell viability tests, and survival analysis. Then, RNA immunoprecipitation was conducted to evaluate the interaction between DDX11-AS1 and RNA-binding protein LIN28A. The regulation effect of LIN28A on autophagy-related genes ATG7 or ATG12 was detected by RNA stability assay and Western blot. Their correlation analysis was evaluated in GEO datasets and further validated by immunohistochemical results. The clinical significance of DDX11-AS1, ATG7, or ATG12 was evaluated by Kaplan-Meier Plotter analysis. Results: Here, we reported DDX11-AS1 was significantly upregulated in chemoresistant breast cancer cells and overexpression of DDX11-AS1 promoted ADR resistance in breast cancer. LIN28A could interact with DDX11-AS1 and was involved in DDX11-AS1-mediated ADR resistance. Interfering with LIN28A reversed DDX11-AS1-induced ADR resistance. LIN28A could increase the protein level of ATG7 and ATG12 by increasing their mRNA stability. Survival analysis showed that ATG12 expression level was negatively correlated with the prognosis of breast cancer patients. Conclusion: This study clarifies the role of DDX11-AS1 in breast cancer chemoresistance and revealed a new mechanism, that is, interacting with LIN28A to stabilize ATG7 and ATG12 and jointly promote chemorefractory. These findings warrant further in vivo investigations to study DDX11-AS1 as a potential target to overcome chemoresistance.