Levels Of Autophagy-related Proteins Research Articles

Ghrelin alleviates hypoxia/reoxygenation-induced H9C2 injury by activating autophagy and AMPK/ULK1 pathway.

The research aims to explore the protective effects of ghrelin and its underlying molecular mechanisms in an H9C2 hypoxia/reoxygenation model. H9C2 cells were transfected with ghrelin overexpression lentiviral vector. The hypoxia/reoxygenation H9C2 model was constructed. The expression of ghrelin was analyzed by qRT-PCR and Western Blotting. CCK8, flow cytometry and TUNEL assay were used to analyze the impact of ghrelin on the survival and apoptosis of H9C2 injured by hypoxia/reoxygenation. The levels of autophagy-related proteins in H9C2 cells were evaluated through Western blotting. ELISA was utilized to assess how ghrelin affects the inflammatory response triggered by hypoxia/reoxygenation. Western blotting was utilized to investigate the regulatory role of ghrelin on the AMPK/ULK1 pathway. Additionally, the AMPK inhibitor Compound C was introduced to delve further into the associated mechanism. Hypoxia/reoxygenation injury decreased the expression of ghrelin. Transfection of ghrelin overexpression lentiviral vector significantly increased the expression of ghrelin in H9C2 cells. Ghrelin overexpression can significantly promote cell survival, reduce apoptosis, activate AMPK, ULK1 and AMBRA1, promote autophagy, increase the expression of LC3BII/LC3BI and Beclin-1, reduce the expression of P62, and reduce inflammatory response. Ghrelin inhibited apoptosis of H9C2 caused by hypoxia/reoxygenation and reduced inflammatory response, which mechanism is related to activation of AMPK/ULK1 pathway and autophagy.

Peroxisome Proliferator-Activated Receptor γ Regulates Lipid Metabolism in Sheep Trophoblast Cells through mTOR Pathway-Mediated Autophagy.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a key nuclear receptor transcription factor that is highly expressed in trophoblastic cells during embryonic attachment and is accompanied by rapid cell proliferation and increased lipid accumulation. We previously showed that the autophagy pathway is activated in cells after activation of PPARγ, accompanied by increased lipid accumulation. In this study, we used PPARγ agonist rosiglitazone and inhibitor GW9662, as well as autophagy activator rapamycin and inhibitor 3-methyladenine, to unravel the probable mechanism of PPARγ engaged in lipid metabolism in sheep trophoblast cells (STCs). After 12 h, 24 h, and 48 h of drug treatment, the levels of autophagy-related proteins were detected by Western blot, the triglyceride content and MDA level of cells were detected by colorimetry, and the lipid droplets and lysosomes were localized by immunofluorescence. We found that PPARγ inhibited the activity of mammalian target of rapamycin (mTOR) pathway in STCs for a certain period of time, promoted the increase of autophagy and lysosome formation, and enhanced the accumulation of lipid droplets and triglycerides. Compared with cells whose PPARγ function is activated, blocking autophagy before activating PPARγ will hinder lipid accumulation in STCs. Pretreatment of cells with rapamycin promoted autophagy with results similar to rosiglitazone treatment, while inhibition of autophagy with 3-methyladenine reduced lysosome and lipid accumulation. Based on these observations, we conclude that PPARγ can induce autophagy by blocking the mTOR pathway, thereby promoting the accumulation of lipid droplets and lysosomal degradation, providing an energy basis for the rapid proliferation of trophoblast cells during embryo implantation. In brief, this study partially revealed the molecular regulatory mechanism of PPARγ, mTOR pathway, and autophagy on trophoblast cell lipid metabolism, which provides a theoretical basis for further exploring the functional regulatory network of trophoblast cells during the attachment of sheep embryos.

HIF-1α-Dependent Autophagy Deficiency in Bone Marrow Endothelial Progenitor Cells Contributes to Acute Graft-Versus-Host Disease after Allotransplant

Background: Acute Graft-Versus-Host Disease (aGVHD) constitutes a prevalent component after allogeneic hematopoietic stem cell transplantation(allo-HSCT). Among the components of the Bone Marrow（BM)microenvironment, Endothelial Progenitor Cells (EPCs) hold significant importance in hematopoietic-supporting and immune reconstitution in post-transplantation patients. Consequently, a discernible correlation emerges between the impairment of EPCs and the incidence and severity of GVHD. Our recent study has shown impaired EPC function and heightened reactive oxygen species (ROS) levels in aGVHD patients (2018, BJH). Additionally, reduced levels of hypoxia-inducible factor-1α (HIF-1α) and autophagy were observed in BM EPCs from aGVHD patients in comparison to those without GVHD（non-GVHD）. HIF-1α is closely linked to ROS generation. Therefore， exploring the role of HIF-1α and autophagy in the development of aGVHD after allo-HSCT is significant. Aims： To conduct a comparative analysis of the functions of EPCs and assess the variations in the expression levels of HIF-1α and autophagy-related proteins between aGVHD and non-GVHD patients. Additionally, the study aims to assess whether the low HIF-1α level is responsible for the autophagy deficiency and mitochondrial dysfunction observed in EPCs of aGVHD patients, and to delineate the specific pathways through which this downregulation influences their capacity of hematopoiesis-supporting. Methods: The current prospective clinical case-matched study included 20 patients with aGVHD and 40 patients without GVHD. BM mononuclear cells（BMMNCs） were cultured as former publications reported. EPCs（CD34+, CD133+, CD309+) were flow-sorted for evaluating apoptosis, ROS, and autophagy levels. SiRNA transfection knocked down HIF-1α in human umbilical vein endothelial cells (HUVECs). Endothelial cells(ECs） gene expression levels were determined using Western blot, RT-qPCR. ECs function was assessed by angiogenesis, Transwell cell migration, CCK-8 cell proliferation, and JC-1 mitochondrial staining. ECs were co-cultured with hematopoietic stem cells （HSCs） for seven days, and HSCs were subjected to colony-forming unit assays and other experiments to evaluate hematopoietic stem cell function. ECs NAD+ and NADH levels were assessed by the kit. Results: Downregulation of autophagy signaling pathway in HUVECs following knockdown of HIF-1α, leading to inhibited cell proliferation, migration, and angiogenesis. Similarly, patients affected by aGVHD exhibited reduced HIF-1α expression, impaired autophagy, and cellular function in EPCs compared to non-GVHD patients. HSCs co-culturing with HUVECs with HIF-1α knockdown and EPCs from aGVHD patients resulted in increased apoptosis, elevated levels of ROS, and impaired colony formation ability. However, intervention with HIF-1α activators Roxadustat, which served to upregulate HIF-1α levels in ECs, activated autophagy, and improved cellular function, also enhanced the hematopoiesis-supporting ability. Furthermore, the knockdown of HIF-1α in HUVECs and EPCs of aGVHD patients led to the abnormal activation of SIRT1 and PARP1 and other NAD+-consuming proteins, which subsequently caused a decrease in NAD+ levels and resulted in abnormal mitochondrial membrane potential. The supplementation of NAD+ precursors, specifically β-nicotinamide mononucleotide, effectively increased NAD+ levels in ECs and restored cellular function. Conclusion: The results revealed that the autophagy deficiency was mediated by reduced expression of HIF-1α in EPCs of patients with aGVHD. The deficiency of autophagy also led to abnormal activation of SIRT1 and PARP1, which in turn affected the levels of NAD+ and mitochondrial membrane potential, ultimately impairing cellular function and hematopoiesis-supporting ability. These molecular changes may be identified as potential contributors to the pathogenesis of aGVHD after allo-HSCT. On the other hand, Roxadustat upregulates HIF-1α expression in ECs, which subsequently enhanced the autophagy level and improved mitochondrial function. As a result, cellular function and hematopoiesis-supporting ability were restored. This promising therapeutic approach highlights a novel strategy for preventing and treating of patients with aGVHD after allo-HSCT.

Influence of Oxa-Nano-Liposome on the Drug Resistance of Gastric Cancer Cells Under p53-Mediated Autophagy

The influence of oxaliplatin (Oxa)-nano-liposomes on the drug resistance of gastric cancer cells (GCCs) and the role of p53-mediated autophagy in this process were investigated in this research. Oxa-nano-liposomes were prepared and their quality was evaluated. GCCs treated with Oxa-nano-liposomes were selected and rolled into a negative control (NC) group (cells+ culture medium), a positive control (PC) group (standard Oxa-nano-liposome), and a Oxa-nano-liposome sample group. Cell inhibition rates (IRs) at changeable drug concentrations (DCs) were compared and analyzed. Furthermore, levels of p53 and autophagy-related proteins (ARPs) (such as LC3-II and p62) in the cells were assessed using Western blotting. The results indicated that Oxa-nano-liposomes prepared (Oxa):natural soy phospholipids (NSP):cholesterol:polyethylene glycol (PEG) 2000 = 1:2:1:1 exhibited the best performance. The Oxa-nano-liposome sample group exhibited a higher cell IR to the NC group, showing a great difference (P <0.05). Additionally, the Oxa-nano-liposome sample group demonstrated superior efficacy compared to the PC group. With increasing DC, p53 and LC3-II were upshifted, while p62 was downshifted. In conclusion, Oxa-nano-liposomes effectively inhibited the growth of GCCs, exhibited improved efficacy, and contributed to reducing drug resistance in GCCs towards Oxa-nano-liposomes. Therefore, the Oxa-nano-Liposomes hold significant potential for clinical application. Moreover, p53 regulated the cellular autophagy, enhancing autophagic activity of GCCs.

The environmental enrichment ameliorates chronic cerebral hypoperfusion-induced cognitive impairment by activating autophagy signaling pathway and improving synaptic function in hippocampus

BackgroundChronic cerebral hypoperfusion (CCH) is a frequently observed underlying pathology of both Alzheimer's disease (AD) and vascular dementia (VD), which is a common consequence of cerebral blood flow (CBF) dysregulation. Synaptic damage has been proven as a crucial causative factor for CCH-related cognitive impairment. This study aimed to investigate the neuroprotective impact of environmental enrichment (EE) intervention on CCH-induced synaptic destruction and the consequent cognitive impairment. Furthermore, the underlying mechanism of this neuroprotective effect was explored to provide new insights into therapeutic interventions for individuals suffering from AD or VD. MethodsIn this experiment, all rats were initially acclimatized to a standard environment (SE) for a period of one week. On the seventh day, rats underwent either bilateral common carotid artery occlusion (2VO) surgery or sham surgery (Sham) before being subjected to a four-week procedure of exposure to an EE, except for the control group. During the EE or SE procedure, intraperitoneal injection of chloroquine (CQ) into rats was performed once daily for four weeks. Following this, cognitive function was assessed using the Morris water maze (MWM) test. The synapse ultrastructure was subsequently observed using transmission electron microscopy. Expression levels of autophagy-related proteins (LC3, LAMP1, and P62) and synapse-related proteins (Synapsin I and PSD-95) were detected through Western blotting. Finally, immunofluorescence was used to examine the expression levels of Synapsin I and PSD-95 and the colocalization of LAMP-1 and LC3 in the hippocampus. ResultsAfter undergoing 2VO, rats exposed to SE exhibited cognitive impairment, autophagic dysfunction, and synapse damage. The synapse damage was evidenced by ultrastructural damage and degradation of synapse-related proteins. However, these effects were significantly mitigated by exposure to an EE intervention. Moreover, the intervention led to an improvement in autophagic dysfunction. ConclusionThe study found that EE had a positive impact on CCH-induced synaptic damage. Specifically, EE was found to increase synaptic plasticity-associated proteins and postsynaptic density thickness, while decreasing synaptic space. This multifaceted effect resulted in an amelioration of CCH-induced cognitive impairment. It was shown that this beneficial outcome was mediated via the activation of the autophagy-lysosomal pathway. Overall, the findings suggest that EE may have a therapeutic potential for cognitive impairments associated with CCH through autophagy-mediated synaptic improvement.

Novel TFG mutation causes autosomal-dominant spastic paraplegia and defects in autophagy

BackgroundMutations in the tropomyosin receptor kinase fused (TFG) gene are associated with various neurological disorders, including autosomal recessive hereditary spastic paraplegia (HSP), autosomal dominant hereditary motor and sensory neuropathy with...

Reduced lysosomal activity and increased amyloid beta accumulation in silica-coated magnetic nanoparticles-treated microglia.

Nanoparticles have been used in neurological research in recent years because of their blood-brain barrier penetration activity. However, their potential neuronanotoxicity remains a concern. In particular, microglia, which are resident phagocytic cells, are mainly exposed to nanoparticles in the brain. We investigated the changes in lysosomal function in silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate dye [MNPs@SiO2(RITC)]-treated BV2 murine microglial cells. In addition, we analyzed amyloid beta (Aβ) accumulation and molecular changes through the integration of transcriptomics, proteomics, and metabolomics (triple-omics) analyses. Aβ accumulation significantly increased in the 0.1μg/μl MNPs@SiO2(RITC)-treated BV2 cells compared to the untreated control and 0.01μg/μl MNPs@SiO2(RITC)-treated BV2 cells. Moreover, the MNPs@SiO2(RITC)-treated BV2 cells showed lysosomal swelling, a dose-dependent reduction in proteolytic activity, and an increase in lysosomal swelling- and autophagy-related protein levels. Moreover, proteasome activity decreased in the MNPs@SiO2(RITC)-treated BV2 cells, followed by a concomitant reduction in intracellular adenosine triphosphate (ATP). By employing triple-omics and a machine learning algorithm, we generated an integrated single molecular network including reactive oxygen species (ROS), autophagy, lysosomal storage disease, and amyloidosis. In silico analysis of the single triple omics network predicted an increase in ROS, suppression of autophagy, and aggravation of lysosomal storage disease and amyloidosis in the MNPs@SiO2(RITC)-treated BV2 cells. Aβ accumulation and lysosomal swelling in the cells were alleviated by co-treatment with glutathione (GSH) and citrate. These findings suggest that MNPs@SiO2(RITC)-induced reduction in lysosomal activity and proteasomes can be recovered by GSH and citrate treatment. These results also highlight the relationship between nanotoxicity and Aβ accumulation.

MiR-455-3p mediates PPARα through UBN2 to promote apoptosis and autophagy in acute myeloid leukemia cells

Acute myeloid leukemia (AML) is one of the deadliest hematologic malignancies, and its targeted therapy has developed slowly. The molecular mechanism of the pathophysiology of the disease remains to be clarified. The aim of our study was to probe the specific regulatory mechanism of miR-455-3p in AML. This study measured the levels of miR-455-3p and ubinuclein-2 (UBN2) in AML cell lines, evaluated cell viability with CCK-8, used flow cytometry to estimate the cell cycle and apoptosis, detected cell apoptosis and autophagy-related protein levels by Western blotting, and added 50 μM chloroquine (CQ) to evaluate the relationship between autophagy and AML. In animal experiments, HL-60 cells were injected into male non-obese diabetic/severe combined immunodeficiency disease (NOD/SCID) mice through the tail vein to determine survival time and observe the degree of liver and spleen damage in the mice. miR-455-3p was prominently reduced in the peripheral blood and AML cell lines, and UBN2 showed high expression. The transfected miR-455-3p mimic effectively restrained the activity of AML cells, whereas overexpression of UBN2 or the addition of the autophagy inhibitor CQ reversed the effect of miR-455-3p. The interaction between UBN2 and peroxisome proliferator-activated receptor alpha (PPARα) was confirmed by coimmunoprecipitation, and overexpression of PPARα reversed the promoting effect of UBN2 knockdown on apoptosis and autophagy in AML cells. In conclusion, miR-455-3p mediates PPARα protein expression through UBN2, exacerbating AML cell apoptosis and autophagy. This study found that miR-455-3p plays an important role in AML cell apoptosis and autophagy, which may provide novel insights for the treatment of AML diseases.

The SGLT2 inhibitor empagliflozin attenuates atherosclerosis progression by inducing autophagy.

Cardiovascular disease due to atherosclerosis is one of the leading causes of death worldwide; however, the underlying mechanism has yet to be defined. The sodium-dependent glucose transporter 2 inhibitor (SGLT2i) empagliflozin is a new type of hypoglycemic drug. Recent studies have shown that empagliflozin not only reduces high glucose levels but also exerts cardiovascular-protective effects and slows the process of atherosclerosis. The purpose of this study was to elucidate the mechanism by which empagliflozin ameliorates atherosclerosis. Male apolipoprotein E-deficient (ApoE-/-) mice were fed a high-fat Western diet to establish an atherosclerosis model. The area and size of atherosclerotic lesions in ApoE-/- mice were then assessed by performing hematoxylin-eosin (HE) staining after empagliflozin treatment. Concurrently, oxidized low-density lipoprotein (oxLDL) was used to mimic atherosclerosis in three different types of cells. Then, following empagliflozin treatment of macrophage cells (RAW264.7), human aortic smooth muscle cells (HASMCs), and human umbilical vein endothelial cells (HUVECs), western blotting was applied to measure the levels of autophagy-related proteins and proinflammatory cytokines, and green fluorescent protein (GFP)-light chain 3 (LC3) puncta were detected using confocal microscopy to confirm autophagosome formation. Oil Red O staining was performed to detect the foaming of macrophages and HASMCs, and flow cytometry was used for the cell cycle analysis. 5-ethynyl-2'-deoxyuridine (EdU), cell counting kit-8 (CCK-8), and scratch assays were also performed to examine the proliferation and migration of HASMCs. Empagliflozin suppressed the progression of atherosclerotic lesions in ApoE-/- mice. Empagliflozin also induced autophagy in RAW246.7 cells, HASMCs, and HUVECs via the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway, and it significantly increased the levels of the Beclin1 protein, the LC3B-II/I ratio, and p-AMPK protein. In addition, empagliflozin decreased the expression of P62 and the protein levels of inflammatory cytokines, and it inhibited the foaming of RAW246.7 cells and HASMCs, as well as the expression of inflammatory factors by inducing autophagy. Empagliflozin activated autophagy through the AMPK signaling pathway to delay the progression of atherosclerosis. Furthermore, the results of flow cytometry, EdU assays, CCK-8 cell viability assays, and scratch assays indicated that empagliflozin blocked HASMCs proliferation and migration. Empagliflozin activates autophagy through the AMPK signaling pathway to delay the evolution of atherosclerosis, indicating that it may represent a new and effective drug for the clinical treatment of atherosclerosis.

Resveratrol Inhibits Abdominal Aortic Aneurysm Progression by Reducing Extracellular Matrix Degradation, Apoptosis, Autophagy, and Inflammation of Vascular Smooth Muscle Cells via Upregulation of HMOX1.

This study aimed to explore the therapeutic effect of resveratrol (RES) against abdominal aortic aneurysm (AAA) and the role of HMOX1 underlying this effect. Vascular smooth muscle cells (VSMCs) were induced by angiotensin II (Ang II) to construct the microenvironment of AAA. HMOX1 expression was downregulated by the short hairpin ribonucleic acid (RNA) specific to HMOX1 in RES-pretreated VSMCs. The levels of matrix metalloproteinase (MMP)-2, MMP-9, and elastin were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot. Apoptosis rate was detected. The levels of apoptosis-related proteins (caspase-3 and Bax/Bcl-2), inflammatory cytokines (interleukin [IL]-6, tumor necrosis factor [TNF]-α, and IL-1β), and autophagy-related proteins (Beclin 1, light chain 3 [LC3] II/I, and p62) were detected by western blot. The secretion of inflammatory factors in cell supernatant was detected by enzyme-linked immunosorbent assay (ELISA). The number of autophagic vesicles in VSMCs was observed and analyzed by transmission electron microscopy. A rat model of pancreatic elastase-induced AAA was established to verify the effect and action mechanism of RES. Stimulation of Ang II increased the messenger RNA (mRNA) and protein levels of MMP-2 and MMP-9, decreased elastin expression, and enhanced apoptosis, secretion of inflammatory factors, and autophagy in VSMCs, whereas RES pretreatment ameliorated Ang II-induced VSMC dysfunction. In addition, HMOX1 mRNA and heme oxygenase-1 (HO-1) protein levels were significantly increased in VSMCs pretreated with RES compared with Ang II treatment alone. Silencing of HMOX1 abolished the effects of RES on VSMC dysfunction. Consistently, RES suppressed the development of AAA in rats by increasing the expression of HMOX1. Resveratrol protects against AAA by inhibiting extracellular matrix degradation, apoptosis, autophagy, and inflammation of VSMCs via HMOX1 upregulation. Our study found that angiotensin II (Ang II) stimulated increased the levels of MMP-2 and MMP-9 in vascular smooth muscle cells (VSMCs), decreased elastin expression, and promoted apoptosis, autophagy occurrence, and secretion of inflammatory factors, while resveratrol (RES) pretreatment improved this effect. In addition, downregulation of HMOX1 expression eliminated the effect of RES on the function of VSMCs. Our study elucidates that RES improves AAA progression through HMOX1 at both cellular and animal levels. This work can help doctors better understand the pathological mechanism of the occurrence and development of AAA, and provide a theoretical basis for clinicians to find better treatment options.

Function and autophagy of monocyte-derived dendritic cells is affected by hepatitis B virus infection

BackgroundThe role of dendritic cells and the autophagy state of dendritic cells in the immune response of hepatitis B virus (HBV) infection was still controversial. In this study, we carefully examined the phenotype, function and autophagy pathway of dendritic cells in HBV infection.MethodsMonocyte-derived dendritic cells from healthy blood donors and patients with chronic HBV infection were stimulated by lipopolysaccharide, supernatant of HepG2.2.15 cells or supernatant of HepG2 cells respectively. Phenotype of dendritic cells was examined by flow cytometry and cytokines secretion was detected by enzyme-linked immunosorbent assay. Autophagy related proteins were detected by western blot and immunofluorescence analysis.ResultsOur results showed that the expression of both major histocompatibility complex II molecules and co-stimulated molecules including cluster of differentiation antigen 80, cluster of differentiation antigen 86 in the monocyte-derived dendritic cells from patients with chronic HBV infection was significantly higher than that from healthy donors when cultured with supernatant of HepG2.2.15 cells. The amount of cytokines, including tumour necrosis factor-α, interleukin-10 and interleukin-12, secreted by monocyte-derived dendritic cells from patients with chronic HBV infection was also significantly higher than that from healthy donors when stimulate by HBV. Interestingly, the expression level of autophagy-related proteins including autophagy-related protein5 and associated protein 1 light chain in dendritic cells from patients with chronic HBV infection was significantly increased when compared with that from healthy donors when re-exposed to HBV.ConclusionsOur results indicated that dendritic cells from patients with chronic HBV infection could intensively present antigen and express co-stimulatory molecules. The increased activation of dendritic cells might be related to the enhanced autophagy of dendritic cells in HBV infection.

Combined Treatment with Bojungikgi-tang (Buzhong Yiqi Decoction) and Riluzole Attenuates Cell Death in TDP-43-Expressing Cells.

To examine the effect of combined treatment with Bojungikgi-tang (BJIGT, Buzhong Yiqi Decoction) and riluzole (RZ) in transactive response DNA-binding protein 43 (TDP-43) stress granule (SG) cells, a amyotrophic lateral sclerosis (ALS) cell line using transcriptomic and molecular techniques. TDP-43 SG cells were pretreated with BJIGT (100 µg/mL), RZ (50 µmol/L), and combined BJIGT (100 µg/mL)/RZ (50 µmol/L) for 6 h before treatment with lipopolysaccharide (LPS, 200 µmol/L). Cell viability assay was performed to elucidate cell toxicity in TDP-43 SC cells using a cell-counting kit-8 (CCK8) assay kit. The expression levels of cell death-related proteins, including Bax, caspase 1, cleaved caspase 3 and DJ1 in TDP-43 SG cells were examined by Western blot analysis. The autophagy-related proteins, including pmTOR/mTOR, LC3b, P62, ATG7 and Bcl-2-associated athanogene 3 (Bag3) were investigated using immunofluorescence and immunoblotting assays. Cell viability assay and Western blot analysis showed that combined treatment with BJIGT and RZ suppressed LPS-induced cell death and expression of cell death-related proteins, including Bax, caspase 1, and DJ1 (P<0.05 or P<0.01). Immunofluorescence and immunoblotting assays showed that combined treatment with BJIGT and RZ reduced LPS-induced formation of TDP-43 aggregates and regulated autophagy-related protein levels, including p62, light chain 3b, Bag3, and ATG7, in TDP-43-expressing cells (P<0.05 or P<0.01). The combined treatment of BJIGT and RZ might reduce inflammation and regulate autophagy dysfunction in TDP-43-induced ALS.

Increased DNMT1 Involvement in the Activation of LO2 Cell Death Induced by Silver Nanoparticles via Promoting TFEB-Dependent Autophagy.

The accumulation of exogenous silver nanoparticles (AgNPs) will terminally bring about liver injury, including cell death, where DNA methylation tends to be a crucial epigenetic modulator. The change in the cell autophagy level verified to be closely associated with hepatocyte death has been followed with wide interest. But the molecular toxicological mechanisms of AgNPs in relation to DNA methylation, autophagy, and cell death remain inconclusive. To address the issue above, in LO2 cells treated with increasing concentrations of AgNPs (0, 5, 10, and 20 μg/mL), a cell cytotoxicity assay was performed to analyze the level of cell death, which also helped to choose an optimal concentration for next experiments. An immunofluorescence assay was used to determine the autophagic flux as well as TFEB translocation, with qRT-PCR and western blot being used to analyze the expression level of autophagy-related genes and proteins. According to our findings, in the determination of cell viability, 20 μg/mL (AgNPs) was adopted as the best working concentration. LO2 cell death, autophagy, and TFEB nuclear translocation were induced by AgNPs, which could be inhibited by lysosome inhibitor chloroquine (CQ) or siRNA specific for TFEB. Moreover, AgNP exposure led to DNA hypermethylation, with DNMT1 taking part mainly, which could be obviously prevented by 5-Aza-2'-deoxycytidine (5-AzaC) or trichostatin A (TSA) treatment or DNMT1 knockout in LO2 cells. Our studies suggest that through TFEB-dependent cell autophagy, increased DNMT1 may facilitate cell death induced by AgNPs.

A new insight of cadmium-induced cellular evidence of autophagic-associated spermiophagy during spermatogenesis.

Autophagy plays a dynamic role in spermatozoa development during spermatogenesis. However, the disruption of autophagic flux induces cell death under metal toxicity and severe oxidative stress. Therefore, we hypothesized that cadmium-induced autophagy might be involved in this mechanism. To verify this hypothesis, we studied cadmium-induced cellular evidence of autophagic-associated spermiophagy within the testis. In the present study, treatment with cadmium caused nuclear depressive disorders and vacuolated mitochondrial damage of Sertoli cells. In addition, spermiophagy through the cellular evidence of spermatozoa phagocytosis, the high lysosomal activity (lysosome engulfment and phagolysosome), and autophagy activity (autolysosome and autophagosome) were observed in the Sertoli cells. The immunohistochemistry of lysosomal membrane protein (LAMP2) to target the phagocytosis of spermatozoa revealed that the immunoreactivity of LAMP2 was overstimulated in the luminal compartment of testis's seminiferous tubules. In addition, the immunohistochemistry and immunofluorescence of autophagy-related protein and microtubule-associated light chain (LC3) results showed the strong immunoreactivity and immunosignaling of LC3 in the Sertoli cells of the testis. Moreover, cadmium caused the overactivation of the expression level of autophagy-related proteins, autophagy-related gene (ATG7), (ATG5), beclin1, LC3, sequestosome 1 (P62), and LAMP2 which were confirmed by western blotting. In summary, this study demonstrated that hazards related to cadmium-induced autophagic-associated spermiophagy with the disruption of autophagic flux, providing new insights into the toxicity of cadmium in mammals and representing a high risk to male fertility.

Bidirectional negative feedback actions of DNMT3A and miR-145 in regulating autophagy in cardiac fibroblasts and affecting myocardial fibrosis.

Epigenetic regulation has crucial implications for myocardial fibrosis. It has been reported that autophagy, regulated by miR-145, is implicated in the proliferation and fibrosis of cardiac fibroblasts (CFs). However, how it works during the process remains unclear. This study explored the exact effects of epigenetic regulation of miR-145 expression on autophagy, proliferation, and fibrosis of CFs. To examine the expression levels of myocardial fibrosis markers (α-SMA and collagen I), autophagy-related proteins (LC3I, LC3II, p62), DNMT3A, and miR-145, qRT-PCR and western blot were employed. And the proliferation of CFs was detected by CCK-8 and ErdU. As for the determination of the binding relationship between DNMT3A and miR-145, dual-luciferase assay was conducted. Next, the detection of the methylation level of the pre-miR-145 promoter region was completed by MSP. And the verification of the effect of the DNMT3A/miR-145 axis on myocardial fibrosis was accomplished by constructing mouse myocardial infarction (MI) models based on the ligation of the left anterior descending method. In TGF-β1-activated CFs, remarkable up-regulation of DNMT3 and considerable down-regulation of miR-145 were observed. And further experiments indicated that DNMT3A was able to down-regulate miR-145 expression by maintaining the hypermethylation level of the pre-miR-145 promoter region. In addition, DNMT3A expression could be directly targeted and negatively modulated by miR-145. Moreover, in vitro cell experiments and mouse MI models demonstrated that DNMT3A overexpression could inhibit autophagy, and promote cell proliferation and fibrosis of CFs. However, this kind of effect could be reversed by miR-145 overexpression. In summary, myocardial fibroblast autophagy can be regulated by bidirectional negative feedback actions of DNMT3A and miR-145, thus affecting myocardial fibrosis. This finding will provide a potential target for the clinical treatment of myocardial fibrosis.

Protective Effect and Mechanism of Autophagy in Endothelial Cell Injury Induced by Hyperoxia.

Bronchopulmonary dysplasia is a chronic lung disease in premature infants with alveolar simplification and pulmonary vascular development disorder as the main pathological feature and hyperoxia as the main etiology. Autophagy is a highly conserved cytological behavior of self-degrading cellular components and is accompanied by oxidative stress. Studies have reported that autophagy is regulated by FOXO1 posttranslational modification. However, whether autophagy can be involved in the regulation of endothelial cell injury induced by hyperoxia and its mechanism are still unclear. We have activated and inhibited autophagy in human umbilical vein endothelial cells under hyperoxia and verified the role of autophagy in endothelial cell-related functions from both positive and negative aspects. Our research showed that the expression level of autophagy-related proteins decreased, accompanied by decreased cell migration ability and tube formation ability and increased cell reactive oxygen species level and cell permeability under hyperoxia conditions. Using an autophagy agonist alleviated hyperoxia-induced changes and played a protective role. However, inhibition of autophagy aggravated the cell damage induced by hyperoxia. Moreover, the decrease in autophagy proteins was accompanied by the upregulation of FOXO1 phosphorylation and acetylation. We concluded that autophagy was a protective mechanism against endothelial cell injury caused by hyperoxia. Autophagy might participate in this process by coregulating posttranslational modifications of FOXO1. · Hyperoxia induces vascular endothelial cell injury.. · Autophagy may has a protective role under hyperoxia conditions.. · FOXO1 posttranslational modification may be involved in the regulation of autophagy..

COTE-1 promotes the proliferation and invasion of small cell lung cancer by regulating autophagy activity via the AMPK/mTOR signaling pathway

BackgroundCOTE-1 has been found to promote the proliferation and invasion of non-small cell lung cancer. However, the mechanism of COTE-1 in SCLC is still unclear. Exploring the role of COTE-1 in SCLC is expected to provide a potential target for the prognosis and treatment of SCLC. MethodsThe expression of COTE-1 and ki-67 was detected by immunohistochemical staining. PCR detected COTE-1 expression level. Cell proliferation activity was detected by CCK8 assay. A wound healing test detected cell migrative ability. Transwell invasion assay detected cell invasive ability. The numbers of autophagosomes were observed by transmission electron microscopy. WB detected the expression levels of autophagy-related proteins and AMPK/mTOR pathway-related proteins. The effect of COTE-1 expression level on the proliferation of SCLC tumor tissues was investigated by establishing a mouse SCLC xenograft tumor model. ResultsThe expression of COTE-1 in SCLC tissues and cells was higher than that in normal tissues and cells. In SCLC cells with high COTE-1 expression, the expression level of autophagy proteins was notably increased, the number of intracellular autophagosomes increased, and the proliferative activity, migration and invasion abilities were enhanced. COTE-1 promotes autophagy, proliferation, and invasion of SCLC cells under nutrient deprivation by activating the AMPK/mTOR signaling pathway. Activation of autophagy by COTE-1 promotes the proliferation and development of xenograft tumors in a mouse model of SCLC. ConclusionCOTE-1 promotes the proliferation, migration and invasion of small cell lung cancer by mediating autophagy based on the AMPK/mTOR pathway.

Seawater pearl hydrolysate inhibits photoaging via decreasing oxidative stress, autophagy and apoptosis of Ultraviolet B-induced human skin keratinocytes.

Ultraviolet (UV) is the main reason to cause photoaging skin which not only hinders beauty, brings the patients with psychological burden, but also pathologically leads to the occurrence of tumors in skin. This study goes into the inhibitory effect and mechanism of seawater pearl hydrolysate (SPH) to address human skin keratinocytes photoaging induced by UVB. The photoaging model of Hacat cell was constructed by UVB irradiation, the levels of oxidative stress, apoptosis, aging, autophagy and autophagy-related protein and signal pathway expression were assessed to characterize the inhibitory effect and mechanism of SPH on photoaging Hacat cell. Seawater pearl hydrolysate significantly accelerated (p < 0.05) the activities of superoxide dismutase, catalase, and glutathione peroxidase, and markedly reduced (p < 0.05) the contents of reactive oxygen species (ROS), malondialdehyde, protein carbonyl compound and nitrosylated tyrosine protein, aging level, apoptosis rate in Hacat cell induced by 200 mJ cm-2 UVB after 24 and 48 h of culture; high dose SPH significantly raised (p < 0.05) relative expression level of p-Akt, p-mTOR proteins, and markedly decreased (p < 0.05) relative expression level of LC3II protein, p-AMPK, and autophagy level in Hacat cell induced by 200 mJ cm-2 UVB, or in combination with the intervention of PI3K inhibitor or AMPK overexpression after 48 h of culture. Seawater pearl hydrolysate can effectively inhibit 200 mJ cm-2 UVB-induced photoaging of Hacat cells. The mechanism indicates removing the excessive ROS through increasing the antioxidation of photoaging Hacat cells. Once redundant ROS is eliminated, SPH works to reduce AMPK, increase PI3K-Akt pathway expression, activate mTOR pathway to lowdown autophagy level, and as a result, inhibit apoptosis and aging in photoaging Hacat cells.

Dinotefuran exposure induces autophagy and apoptosis through oxidative stress in Bombyx mori

As a third-generation neonicotinoid insecticide, dinotefuran is extensively used in agriculture, and its residue in the environment has potential effects on nontarget organisms. However, the toxic effects of dinotefuran exposure on nontarget organism remain largely unknown. This study explored the toxic effects of sublethal dose of dinotefuran on Bombyx mori. Dinotefuran upregulated reactive oxygen species (ROS) and malondialdehyde (MDA) levels in the midgut and fat body of B. mori. Transcriptional analysis revealed that the expression levels of many autophagy and apoptosis-associated genes were significantly altered after dinotefuran exposure, consistent with ultrastructural changes. Moreover, the expression levels of autophagy-related proteins (ATG8-PE and ATG6) and apoptosis-related proteins (BmDredd and BmICE) were increased, whereas the expression level of an autophagic key protein (sequestosome 1) was decreased in the dinotefuran-exposed group. These results indicate that dinotefuran exposure leads to oxidative stress, autophagy, and apoptosis in B. mori. In addition, its effect on the fat body was apparently greater than that on the midgut. In contrast, pretreatment with an autophagy inhibitor effectively downregulated the expression levels of ATG6 and BmDredd, but induced the expression of sequestosome 1, suggesting that dinotefuran-induced autophagy may promote apoptosis. This study reveals that ROS generation regulates the impact of dinotefuran on the crosstalk between autophagy and apoptosis, laying the foundation for studying cell death processes such as autophagy and apoptosis induced by pesticides. Furthermore, this study provides a comprehensive insight into the toxicity of dinotefuran on silkworm and contributes to the ecological risk assessment of dinotefuran in nontarget organisms.

α-amanitin induces autophagy through AMPK-mTOR-ULK1 signaling pathway in hepatocytes

Amanitin poisoning is one of the most life-threatening mushroom poisonings. α-Amanitin plays a key role in Amanita phalloides intoxication. α-Amanitin shows toxic effects on the liver. However, the mechanism by which α-amanitin induces liver injury has not been elucidated. Autophagy plays a crucial role in maintaining cellular homeostasis and is closely related to the occurrence of a variety of diseases. Studies have shown that autophagy may play an important role in the process of α-amanitin-induced liver injury. However, the mechanism of α-amanitin-induced autophagy remains unclear. Thus, this study aimed to explore the mechanisms of α-amanitin in inducing hepatotoxicity in Sprague Dawley (SD) rats and the normal human liver cell line L02 cells. The SD rats and L02 cells exposed to α-amanitin were observed to determine whether α-amanitin could induce the autophagy of rat liver and L02 cells. The regulatory relationship between autophagy and the AMPK-mTOR-ULK pathway by exposing the autophagy agonist (rapamycin (RAPA)), autophagy inhibitor (3-methylademine (3-MA)), and AMPK inhibitor (compound C) was also explored. Autophagy-related proteins and AMPK-mTOR-ULK pathway-related proteins were detected using Western blot. The results of the study indicated that exposure to different concentrations of α-amanitin led to morphological changes in liver cells and significantly elevated levels of ALT and AST in the serum of SD rats. Additionally, the expression levels of LC3-II, Beclin-1, ATG5, ATG7, AMPK, p-AMPK, mTOR, p-mTOR, and ULK1 were significantly increased in the rat liver. And we found that L02 cells exposed to 0.5 μM α-amanitin for 6 h significantly induced autophagy and activated the AMPK-mTOR-ULK1 pathway. Pretreated with RAPA, 3-MA, and compound C for 1 h, the expression levels of autophagy-related proteins and AMPK-mTOR-ULK pathway-related proteins significantly changed. Our results indicates that autophagy and the AMPK-mTOR-ULK pathway are involved in the process of α-amanitin-induced liver injury. This study may foster the identification of actionable therapeutic targets for A. phalloides intoxication.