STM2457 Inhibits METTL3-Mediated m6A Modification of miR-30c to Alleviate Spinal Cord Injury by Inducing the ATG5-Mediated Autophagy.

OBJECTIVE: The study aimed to investigate the role of N6-methyladenosine (m6A) modification in spinal cord injury (SCI) and its underlying mechanism, focusing on the interplay between m6A methyltransferase-like 3 (METTL3), miR-30c, and autophagy-related proteins. METHODS: An SCI model was established in rats, and changes in autophagy-related proteins, m6A methylation levels, and miR-30c levels were analyzed. Hydrogen peroxide (H2O2)-stimulated spinal cord neuron cells (SCNCs) were used to assess the impact of METTL3 overexpression. The effects of STM2457, an antagonist of METTL3, were evaluated on cell viability, apoptosis, and autophagy markers in H2O2-stimulated SCNCs. RESULTS: In the SCI model, decreased levels of autophagy markers and increased m6A methylation, miR-30c levels, and METTL3 were observed. Overexpression of METTL3 in SCNCs led to reduced cell viability, increased apoptosis, and suppressed autophagy. Conversely, co-overexpression of autophagy-related protein 5 (ATG5) or miR-30c inhibition reversed these effects. Knocking out METTL3 yielded opposite results. STM2457 treatment improved cell viability, reduced apoptosis, and upregulated autophagy markers in SCNCs, which also enhanced functional recovery in rats as measured by the Basso-Beattie-Bresnahan score and inclined plate test. CONCLUSION: STM2457 alleviated SCI by suppressing METTL3-mediated m6A modification of miR-30c, which in turn induces ATG5-mediated autophagy. This study provides insights into the role of m6A modification in SCI and suggests a potential therapeutic approach through targeting METTL3.

PEI/MMNs@LNA-542 nanoparticles alleviate ICU-acquired weakness through targeted autophagy inhibition and mitochondrial protection.

Intensive care unit-acquired weakness (ICU-AW) is prevalent in critical care, with limited treatment options. Certain microRNAs, like miR-542, are highly expressed in ICU-AW patients. This study investigates the regulatory role and mechanisms of miR-542 in ICU-AW and explores the clinical potential of miR-542 inhibitors. ICU-AW models were established in C57BL/6 mice through cecal ligation and puncture (CLP) and in mouse C2C12 myoblasts through TNF-α treatment. In vivo experiments demonstrated decreased muscle strength, muscle fiber atrophy, widened intercellular spaces, and increased miR-542-3p/5p expression in ICU-AW mice model. In vitro experiments indicated suppressed ATG5, ATG7 and LC3II/I, elevated MDA and ROS levels, decreased SOD levels, and reduced MMP in the model group. Similar to animal experiments, the expression of miR-542-3p/5p was upregulated. Gel electrophoresis explored the binding of polyethyleneimine/mesoporous silica nanoparticles (PEI/MMNs) to locked nucleic acid (LNA) miR-542 inhibitor (LNA-542). PEI/MMNs@LNA-542 with positive charge (3.03 ± 0.363 mV) and narrow size (206.94 ± 6.19 nm) were characterized. Immunofluorescence indicated significant internalization with no apparent cytotoxicity. Biological activity, examined through intraperitoneal injection, showed that PEI/MMNs@LNA-542 alleviated muscle strength decline, restored fiber damage, and recovered mitochondrial injury in mice. In conclusion, PEI/MMNs nanoparticles effectively delivered LNA-542, targeting ATG5 to inhibit autophagy and alleviate mitochondrial damage, thereby improving ICU-AW.

Macrophage autophagy protects against acute kidney injury by inhibiting renal inflammation through the degradation of TARM1.

Macroautophagy/autophagy activation in renal tubular epithelial cells protects against acute kidney injury (AKI). However, the role of immune cell autophagy, such as that involving macrophages, in AKI remains unclear. In this study, we discovered that macrophage autophagy was an adaptive response during AKI as mice with macrophage-specific autophagy deficiency (atg5-/-) exhibited higher serum creatinine, more severe renal tubule injury, increased infiltration of ADGRE1/F4/80+ macrophages, and elevated expression of inflammatory factors compared to WT mice during AKI induced by either LPS or unilateral ischemia-reperfusion. This was further supported by adoptive transfer of atg5-/- macrophages, but not WT macrophages, to cause more severe AKI in clodronate liposomes-induced macrophage depletion mice. Similar results were also obtained in vitro that bone marrow-derived macrophages (BMDMs) lacking Atg5 largely increased pro-inflammatory cytokine expression in response to LPS and IFNG. Mechanistically, we uncovered that atg5 deletion significantly upregulated the protein expression of TARM1 (T cell-interacting, activating receptor on myeloid cells 1), whereas inhibition of TARM1 suppressed LPS- and IFNG-induced inflammatory responses in atg5-/- RAW 264.7 macrophages. The E3 ubiquitin ligases MARCHF1 and MARCHF8 ubiquitinated TARM1 and promoted its degradation in an autophagy-dependent manner, whereas silencing or mutation of the functional domains of MARCHF1 and MARCHF8 abolished TARM1 degradation. Furthermore, we found that ubiquitinated TARM1 was internalized from plasma membrane into endosomes, and then recruited by the ubiquitin-binding autophagy receptors TAX1BP1 and SQSTM1 into the autophagy-lysosome pathway for degradation. In conclusion, macrophage autophagy protects against AKI by inhibiting renal inflammation through the MARCHF1- and MARCHF8-mediated degradation of TARM1.Abbreviations: AKI, acute kidney injury; ATG, autophagy related; Baf, bafilomycin A1; BMDMs, bone marrow-derived macrophages; CCL2/MCP-1, C-C motif chemokine ligand 2; CHX, cycloheximide; CQ, chloroquine; IFNG, interferon gamma; IL, interleukin; IR, ischemia-reperfusion; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; LPS, lipopolysaccharide; MARCHF, membrane associated ring-CH-type finger; NC, negative control; NFKB, nuclear factor of kappa light polypeptide gene enhancer in B cells; NLRP3, NLR family, pyrin domain containing 3; NOS2, nitric oxide synthase 2, inducible; Rap, rapamycin; Wort, wortmannin; RT-qPCR, real-time quantitative polymerase chain reaction; Scr, serum creatinine; SEM, standard error of mean; siRNA, small interfering RNA; SYK, spleen tyrosine kinase; TARM1, T cell-interacting, activating receptor on myeloid cells 1; TAX1BP1, Tax1 (human T cell leukemia virus type I) binding protein 1; TECs, tubule epithelial cells; TNF, tumor necrosis factor; WT, wild type.

The RNA N6-methyladenosine demethylase FTO regulates ATG5 to inhibit malignant progression of uveal melanoma.

PURPOSE: This research aimed to identify the function of fat mass- and obesity-associated protein (FTO), an eraser of N6-methyladenosine (m6A), and explore its possible mechanisms in uveal melanoma (UVM). METHODS: We performed quantitative real-time PCR (qPCR), Western blotting and gene correlation analysis with GEPIA2 to assess FTO expression and identify its potential targets in UVM. CCK-8, colony formation, cell cycle, cell apoptosis, wound healing and Transwell invasion assays were utilized to assess cell viability, cell cycle distribution, apoptosis, migration and invasion. Western blotting, qPCR and methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) were carried out to explore the underlying mechanism of FTO in 2 UVM cell lines. RESULTS: FTO, a key m6A demethylase, was found to be upregulated in human UVM tissues compared with normal choroid tissues. Knockdown of FTO in Mel270 and OMM2.3 cells significantly promoted proliferation and migration and suppressed apoptosis. Mechanistically, knockdown of FTO decreased the expression of ATG5, an autophagy-related gene, leading to attenuation of autophagosome formation, thereby inhibiting autophagy. Upon FTO knockdown, increased levels of methylated ATG5 and decreased ATG5 stability were detected. Furthermore, ATG5 dramatically alleviated FTO downregulation-induced tumor growth and metastasis. CONCLUSIONS: Our research highlights the importance of the m6A demethylase FTO in UVM by demonstrating that it direct regulates ATG5-induced autophagy in an m6A-dependent manner. These findings suggest that FTO may serve as a potential therapeutic target for UVM.

USP13 promotes acute myeloid leukemia cell proliferation and autophagy by promoting ATG5.

OBJECTIVE: To elucidate the role of USP13 in acute myeloid leukemia (AML) by investigating its effects on cell growth, apoptosis and autophagy, and to explore the underlying mechanisms. METHODS: The expression of USP13 in AML cells was assessed using quantitative PCR (qPCR) and immunoblotting. Cell Counting Kit-8 (CCK-8) and Edu staining were employed to evaluate the impact of USP13 on AML cell growth. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and immunostaining assays were conducted to examine the effects of USP13 on apoptosis and autophagy in AML cells, and immunoblot assays were performed to determine the potential underlying mechanistic pathway. RESULTS: USP13 expression was significantly elevated in AML cells, correlating with enhanced cell proliferation and resistance to apoptosis. Moreover, USP13 promoted autophagy in AML cells. Mechanistically, USP13 was found to be associated with upregulating ATG5 expression, which promoted AML progression. CONCLUSION: USP13 promotes AML cell growth and autophagy by upregulating ATG5.

The role of alternative autophagy in cell viability and response to paclitaxel treatment in v-Ha-ras-transformed NIH 3T3 cells.

In confluent v-Ha-ras-transformed NIH 3T3 fibroblasts (Ras-NIH 3T3), LC3 downregulation may precede a decrease in canonical autophagy, thus contributing to cell survival. Herein, we aimed to investigate the role of alternative autophagy in the viability of long-term cultures of Ras-NIH 3T3 cells and their parental NIH 3T3 cells. As cell confluence increased with the culture period, the level of alternative autophagy, as assessed through Lamp2-Rab9 co-localization, gradually decreased in both cell lines. However, Ras-NIH 3T3 cells maintained higher levels of alternative autophagy than the parental cells did. Rab9 knockdown minimally affected NIH 3T3 cells while drastically reducing the viability of Ras-NIH 3T3 cells, which suggested that alternative autophagy plays a critical role in Ras-NIH 3T3 cells. In contrast, reactive oxygen species (ROS) production in Ras-NIH 3T3 cells was higher than that in NIH 3T3 cells during long-term culture. Moreover, NIH 3T3 cells exhibited a continual decrease in mitochondrial mass, whereas Ras-NIH 3T3 cells maintained high mitochondrial mass. Immunofluorescence analysis of mitochondrial membrane marker proteins and mitochondrial membrane potential (MMP) also demonstrated a temporal pattern of changes similar to those of mitochondrial mass. This finding could be attributed to the relatively higher level of alternative autophagy in Ras-NIH 3T3 cells facilitating the removal of damaged mitochondria. Paclitaxel treatment in Ras-NIH 3T3 cells induced an increase in canonical autophagy rates along with suppression of alternative autophagy. Ras-NIH 3T3 cells showed high sensitivity to paclitaxel at the early stage of culture, but as cell confluence increased, resistance to paclitaxel increased, showing a similar level of cell viability to the vehicle control group. The study findings suggest that alternative autophagy is more important than canonical autophagy for maintaining cell survival in response to an unfavorable environment, such as during high cell confluence and exposure to anticancer agents.

Increased serum ATG5 as a marker of autophagy in psoriasis vulgaris patients: a cross-sectional study.

Psoriasis (PsO) is a prevalent chronic inflammatory skin disease. It is a complex condition that is affected by environmental and hereditary variables. Numerous pathogens, including viruses, bacteria, and even fungi, have been linked to PsO. One of the mechanisms that clears infections is autophagy. The mechanism by which a cell feeds itself is called autophagy by reusing cytoplasmic components in the lysosome. The autophagy-related (ATG) proteins are essential components of the system that control the strictly regulated process of autophagy. Among these 41 proteins, ATG5 is one that is required in order for autophagic vesicles to develop. This research aimed to compare ATG5 levels in serum among those suffering from psoriasis vulgaris and healthy controls. This cross-sectional research was carried out on 45 individuals with vulgaris psoriasis and 45 healthy, sex and age-matched control subjects. All participants underwent a clinical examination, a laboratory investigation, and a history taking, including lipid profiles and serum ATG5. The mean age of the control and PsO were 40.6 ± 9.6, and 43.7 ± 9.3 years respectively. The mean total PASI score was 13.9 ± 8.9, with a median of 11.7 (8.8). According to the PASI score, about 38% (n = 17) had mild disease (PASI < 10), and about 62% (n = 28) had moderate/severe disease (PASI ≥ 10). There was a significantly higher median (IQR) (25th-75th) ATG5 level in PsO 206 (97) (145-242) ng/ml than in the control 147 (98) (111-209) ng/ml (p = 0.002). An insignificant higher median level (IQR) was observed in PsO with mild disease 207(95) compared with those with moderate/severe disease 183(98.5) (p = 0.057). Dissimilarly, the median (IQR) ATG5 level was significantly lower in PsO individuals with metabolic syndrome 170(72) compared with those without 207(104) (p = 0.044). Four predictors were identified following sex and age adjustments, in the final linear regression model: PASI score, triglyceride, High-Density Lipoprotein, and presence of metabolic syndrome. There can be a connection between autophagy as measured by ATG5 and psoriasis vulgaris. ATG5 was elevated in the serum of individuals with psoriasis vulgaris. However, it decreased in patients with metabolic syndrome. No relation was found between serum ATG5 and PASI score. Psoriasis vulgaris patients may benefit from using an autophagy enhancer as a potential treatment target.

Assessing DNA methylation of ATG 5 and MAP1LC3Av1 gene in oral squamous cell carcinoma and oral leukoplakia- a cross sectional study.

Background: The progression and pathogenesis of oral cancer is greatly impacted by epigenetic modifications, such as DNA methylation. Autophagy, is an adaptive mechanism used to maintain the survival and integrity of cells. Oral squamous cell carcinoma is linked to a number of autophagy indicators, although it is yet unknown if DNA methylation of autophagy-related genes promotes the development of oral leukoplakia (OL), oral squamous cell carcinoma (OSCC). Aim: Our study was aimed to assess, compare and evaluate the DNA methylation of ATG5 and MAP1LC3Av1 genes in oral leukoplakia, oral squamous cell carcinoma. Materials and methods: This cross-sectional study was designed with sample size of 48 tissues which was clinically and histopathologically diagnosed as OL, OSCC and normal tissue. The samples were divided into three groups (Group A, Group B, and Group C; (n = 16 each). Following histopathological confirmation, the tissue was stored in the RNA reagent, then subjected to DNA extraction, methylation-sensitive polymerase chain reaction (MS-PCR). DNA methylation of the ATG5 and MAP1LC3Av1 genes were assessed. Results: Shapiro-Wilk and Kolmogorov-Smirnov tests showed that the values were normally distributed. Both the ATG5 and MAP1LC3Av1 genes were methylated in OSCC, OL tissues compared to normal tissues. A statistically significant results was seen among the three study groups. Conclusion: A significant difference was noted in the hypermethylation status of the promoter regions of the ATG5 and MAP1LC3Av1 genes. This provides some insight into their crucial role in the development of tumors. Future research with larger sample is needed to assess its potential clinical implications in oral carcinoma.

Herpes Simplex Virus type 1 inhibits autophagy in glial cells but requires ATG5 for the success of viral replication.

Herpes Simplex Virus type 1 (HSV-1) 1 is a neurotropic virus that has been associated with neurodegenerative disorders. The dysregulation of autophagy by HSV-1 has been proposed as a potential cause of neurodegeneration. While studies have extensively tackled the interaction between autophagy and HSV-1 in neurons, research in glial cells is currently limited. Our studies demonstrate that HSV-1 inhibits, but not completely blocks, the formation of autophagosomes in human oligodendroglioma- and astrocytoma- derived cell lines. These findings have been confirmed in murine oligodendrocyte precursor cells (OPCs). Finally, this study investigates the impact of autophagy on HSV-1 infection in glial cells. While the lack of basal autophagy in LC3B knockout glial cells does not have a significant effect on viral infection, cells without the autophagy-related protein ATG5 exhibit reduced viral production. The absence of ATG5 leads to a decrease in the transcription and replication of viral genes, as well as a delay in the initial stages of the formation of HSV-1 replication compartments. These findings indicate that while autophagy may not play a significant role in antiviral defense in glial cells, HSV-1 may be inhibiting autophagy to exploit non-canonical functions of certain components of the autophagic machinery, such as ATG5, to benefit its lifecycle.

METTL14 is Involved in TNF-α-Induced Inflammation in Colorectal Epithelial Cells via Autophagy Modulation.

Ulcerative colitis (UC) is a chronic and relapsing inflammatory bowel disease (IBD) characterized by colorectal inflammation. The N6-methyladenosine (m6A) modification of RNA regulates gene expression through the modulation of RNA metabolism, thus influencing various physiological and pathological processes. The aim of this study was to investigate the biological function of m6A methyltransferase METTL14 in colorectal epithelial cell inflammation. Bioinformatics analysis indicated that METTL14 expression was decreased in UC and was associated with disease severity and immune infiltration. We also noted a downregulation of METTL14 expression and a decrease in the total m6A RNA levels in TNF-α-stimulated Caco-2 cells. Moreover, METTL14 knockdown promoted inflammation and inhibited autophagy in TNF-α-stimulated Caco-2 cells, as indicated by the upregulation of NF-κB signaling and pro-inflammatory cytokine expression as well as LC3B protein downregulation. Treatment with the autophagy activator Torin-1 ameliorated the pro-inflammatory effects of METTL14 silencing. Furthermore, METTL14 knockdown significantly reduced the expression of ATG5. ATG5 overexpression could nullify the pro-inflammatory effect of METTL14 knockdown in TNF-α-stimulated Caco-2 cells. Mechanistically, METTL14 knockdown promoted ATG5 mRNA degradation, and luciferase analysis identified ATG5 as a target of m6A modification by METTL14. Taken together, silencing METTL14 promoted inflammation in Caco-2 cells via the downregulation of ATG5. Our findings revealed the importance of the m6A modification in colonic inflammation and autophagy, indicating that targeting METTL14 might be a potential therapeutic strategy for anti-inflammatory treatment in UC.

The role of Atg5 gene in tumorigenesis under autophagy deficiency conditions.

Autophagy is a self-recycling machinery to maintain cellular homeostasis by degrading harmful materials in the cell. Autophagy-related gene 5 (Atg5) is required for autophagosome maturation. However, the role of Atg5 in tumorigenesis under autophagy deficient conditions remains unclear. This study focused on the autophagy-independent role of Atg5 and the underlying mechanism in tumorigenesis. We demonstrated that knockout of autophagy-related genes including Atg5, Atg7, Atg9, and p62 in mouse embryonic fibroblast (MEF) cells consistently decreased cell proliferation and motility, implying that autophagy is required to maintain diverse cellular functions. An Atg7 knockout MEF (Atg7-/- MEF) cell line representing deprivation of autophagy function was used to clarify the role of Atg5 transgene in tumorigenesis. We found that Atg5-overexpressed Atg7-/-MEF (clone A) showed increased cell proliferation, colony formation, and migration under autophagy deficient conditions. Accordingly, rescuing the autophagy deficiency of clone A by overexpression of Atg7 gene shifts the role of Atg5 from pro-tumor to anti-tumor status, indicating the dual role of Atg5 in tumorigenesis. Notably, the xenograft mouse model showed that clone A of Atg5-overexpressed Atg7-/- MEF cells induced temporal tumor formation, but could not prolong further tumor growth. Finally, biomechanical analysis disclosed increased Wnt5a secretion and p-JNK expression along with decreased ß-catenin expression. In summary, Atg5 functions as a tumor suppressor to protect the cell under normal conditions. In contrast, Atg5 shifts to a pro-tumor status under autophagy deprivation conditions.

Metallothionein 3 Potentiates Pulmonary Artery Smooth Muscle Cell Proliferation by Promoting Zinc-MTF1-ATG5 Axis-mediated Autophagosome Formation.

Abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) is one of the critical pathological mechanisms of pulmonary hypertension (PH), and therefore is gradually being adopted as an important direction for the treatment of PH. Metallothioneins (MTs) have been reported to be associated with PH, but the underlying mechanisms are not fully understood. Here, we demonstrated that the expression level of metallothionein 3 (MT3) was significantly increased in pulmonary arterioles from PH patients and chronic hypoxia-induced rat and mouse PH models, as well as in hypoxia-treated human PASMCs. Knockdown of MT3 significantly inhibited the proliferation of human PASMCs by arresting the cell cycle in the G1 phase, while overexpression of MT3 had the opposite effect. Mechanistically, we found that MT3 increased the intracellular zinc (Zn2+) concentration to enhance the transcriptional activity of metal-regulated transcription factor 1 (MTF1), which promoted the expression of autophagy-related gene 5 (ATG5), facilitating autophagosome formation. More importantly, MT3-induced autophagy and proliferation of human PASMCs were largely prevented by knockdown of MTF1 and ATG5. Therefore, in this study, we identified MT3-Zinc-MTF1-ATG5 as a novel pathway that affects PASMC proliferation by regulating autophagosome formation, suggesting that MT3 may be a novel target for the treatment of PH.

Autophagy in hematopoietic stem cell development of the embryo.

Hematopoietic stem cells (HSC) emerge from hemogenic endothelial cells (HEC) in the aorta-gonad-mesonephros (AGM) region of embryos, which go through the pre-HSC process. Various intrinsic and extrinsic factors are involved in this process. We recently discovered that the existence of distinct macroautophagic/autophagic statuses in hematopoietic precursors is related to the hematopoietic potential of pre-HSCs and the depletion of the Atg5 (autophagy related 5) gene specifically in endothelial cells impaired in the transition of endothelial to pre-HSCs, by hampering the autophagic process, likely via the NCL (nucleolin) pathway.Abbreviation: Atg5: autophagy related 5; EGFP: enhanced green fluorescent protein; EHT: endothelial-to-hematopoietic transition; HEC: hemogenic endothelial cell; HSC: hematopoietic stem cell; NCL: nucleolin; RFP: red fluorescent protein.

Autophagy-related protein 5 (ATG5) interacts with bone marrow stromal cell antigen 2 (BST2) to stimulate HBV replication through antagonizing the antiviral activity of BST2.

Hepatitis B virus (HBV) infection is a major global health burden with 820 000 deaths per year. In our previous study, we found that the knockdown of autophagy-related protein 5 (ATG5) significantly upregulated the interferon-stimulated genes (ISGs) expression to exert the anti-HCV effect. However, the regulation of ATG5 on HBV replication and its underlying mechanism remains unclear. In this study, we screened the altered expression of type I interferon (IFN-I) pathway genes using RT² Profiler™ PCR array following ATG5 knock-down and we found the bone marrow stromal cell antigen 2 (BST2) expression was significantly increased. We then verified the upregulation of BST2 by ATG5 knockdown using RT-qPCR and found that the knockdown of ATG5 activated the Janus kinase/signal transducer and activator of transcription (JAK-STAT) signaling pathway. ATG5 knockdown or BST2 overexpression decreased Hepatitis B core Antigen (HBcAg) protein, HBV DNA levels in cells and supernatants of HepAD38 and HBV-infected NTCP-HepG2. Knockdown of BST2 abrogated the anti-HBV effect of ATG5 knockdown. Furthermore, we found that ATG5 interacted with BST2, and further formed a ternary complex together with HBV-X (HBx). In conclusion, our finding indicates that ATG5 promotes HBV replication through decreasing BST2 expression and interacting with it directly to antagonize its antiviral function.

SS-31 inhibits the inflammatory response by increasing ATG5 and promoting autophagy in lipopolysaccharide-stimulated HepG2 cells.

SS-31 is a mitochondria-targeting short peptide. Recent studies have indicated its hepatoprotective effects. In our study, we investigated the impact of SS-31 on LPS-induced autophagy in HepG2 cells. The results obtained from a dual-fluorescence autophagy detection system revealed that SS-31 promotes the formation of autolysosomes and autophagosomes, thereby facilitating autophagic flux to a certain degree. Additionally, both ELISA and qPCR analyses provided further evidence that SS-31 safeguards HepG2 cells against inflammatory responses triggered by LPS through ATG5-dependent autophagy. In summary, our study demonstrates that SS-31 inhibits LPS-stimulated inflammation in HepG2 cells by upregulating ATG5-dependent autophagy.

Indobufen alleviates ischemic stroke injury by regulating transcription factor NRF2 and inhibiting ATG5 expression.

BACKGROUND: Ischemic stroke (IS) is a detrimental neurological disease and IS lacks valuable methods to recover body function. Indobufen (IND) could alleviate IS. However, the possible mechanism remains undefined. METHODS: SH-SY5Y cells were cultured under the oxygen-glucose deprivation/reoxygenation (OGD/R) environment and then were treated with small interfering RNA (siRNA) of NRF2 and ATG5. The influence of various concentrations of IND (50 µM, 100 µM, 200 µM, and 400 µM) was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide. Levels of superoxide dismutase (SOD) and malonaldehyde (MDA) were examined by ELISA. Reactive oxygen species (ROS) production was determined by DCFH-DA staining. The protein levels of LC3II/LC3I, Beclin1, p62, NRF2, and ATG5 were detected by western blot. RESULTS: IND increased cell viability, while depressed the rate of apoptosis in SH-SY5Y cells of OGD/R environment. IND inhibited autophagy by suppressing the levels of LC3II/LC3I, Beclin1 protein, and increasing p62 protein expression in SH-SY5Y cells of OGD/R environment. IND limited the contents of ROS and MDA, while amplifying the activity of SOD in SH-SY5Y cells with OGD/R exposure. IND also promoted NRF2 expression in OGD/R environment. CONCLUSION: IND could inhibit autophagy, oxidative stress, and apoptosis in SH-SY5Y cells with OGD/R exposure, further alleviating IS injury by regulating transcription factor NRF2 and inhibiting ATG5 expression.

The role and mechanism of macrophage autophagy in the experimental model of chronic obstructive pulmonary disease.

INTRODUCTION: Macrophages play an important role in chronic obstructive pulmonary disease (COPD). Cigarette smoke (CS) impairs autophagy in alveolar macrophages from COPD patients, and autophagic impairment leads to reduced clearance of protein aggregates, dysfunctional mitochondria, and defective bacterial delivery to lysosomes. However, the exact function of lung macrophage autophagy in the pathogenesis of CS-induced COPD remains largely unknown. METHODS: Western blot detected the expression of autophagy-related proteins induced by CSE. The model of COPD mice was established by CS exposure combined with CSE intraperitoneal injection. Double immunofluorescence was used to measure the CD206+LC3B+ cells. The morphological changes and effects on lung function were observed. Masson staining detected the changes in collagen fibers in lung tissue. The expression levels of E-cadherinb and N-cadherinb were detected by immunohistochemistry. Western blot detected the expression of ATP6V1E1 in lung tissue. RESULTS: At 24 hours of exposure to CSE, the expression levels of LC3B (microtubule-associated protein 1A/1B-light chain 3B) and P62 (nucleoporin 62) were highest at 1% CSE and AGT5 (nucleoporin 62) at 2.5% CSE; at 48 hours, the expression levels of LC3B, P62 and AGT5 were highest at 2.5% CSE, and as the intervention time increased.CD206+LC3B+ cells were significantly higher in the COPD group. Enhanced macrophage autophagy may promote emphysema formation and aggravate lung function damage. The expression of E-cadherinb in lung tissue of the COPD group was decreased, and N-cadherinb expression was increased; the expression of E-cadherinb was increased, and N-cadherinb expression was decreased in ATG5myeΔ COPD mice. The expression of ATP6V1E1 in the lung tissue was increased in the COPD group; ATP6V1E1 expression was decreased in the lung tissues of ATG5myeΔ COPD mice. CONCLUSIONS: CSE enhanced macrophage autophagy, leads to increased lung function impairment and collagenous fiber in lung tissue, as well as promotes epithelial-mesenchymal transition, and eventually leads to small airway remodeling, which may be achieved through the ATG5/ATP6V1E1 pathway.

Association of Circulating Autophagy Proteins ATG5 and Beclin 1 with Acute Myocardial Infarction in a Case-Control Study.

INTRODUCTION: Acute myocardial infarction (AMI) is a main contributor of sudden cardiac death worldwide. The discovery of new biomarkers that can improve AMI risk prediction meets a major clinical need for the identification of high-risk patients and the tailoring of medical treatment. Previously, we reported that autophagy a highly conserved catabolic mechanism for intracellular degradation of cellular components is involved in atherosclerotic plaque phenotype and cardiac pathological remodeling. The crucial role of autophagy in the normal and diseased heart has been well described, and its activation functions as a pro-survival process in response to myocardial ischemia. However, autophagy is dysregulated in ischemia/reperfusion injury, thus promoting necrotic or apoptotic cardiac cell death. Very few studies have focused on the plasma levels of autophagy markers in cardiovascular disease patients, even though they could be companion biomarkers of AMI injury. The aims of the present study were to evaluate (1) whether variations in plasma levels of two key autophagy regulators autophagy-related gene 5 (ATG5) and Beclin 1 (the mammalian yeast ortholog Atg6/Vps30) are associated with AMI and (2) their potential for predicting AMI risk. METHODS: The case-control study population included AMI patients (n = 100) and control subjects (n = 99) at high cardiovascular risk but without known coronary disease. Plasma levels of ATG5 and Beclin 1 were measured in the whole population study by enzyme-linked immunosorbent assay. RESULTS: Multivariate analyses adjusted on common cardiovascular factors and medical treatments, and receiver operating characteristic curves demonstrated that ATG5 and Beclin 1 levels were inversely associated with AMI and provided original biomarkers for AMI risk prediction. CONCLUSION: Plasma levels of autophagy regulators ATG5 and Beclin 1 represent relevant candidate biomarkers associated with AMI.

ATG5 nonautophagically regulates inflammation and differentiation in mouse embryonic stem cells.

Embryonic stem cells (ESCs), with abilities of infinite proliferation (self-renewal) and to differentiate into distinct cell types (pluripotency), show attenuated inflammatory response against cytokines or pathogens, which is recognized as a unique characteristic of ESCs compared with somatic cells. However, the underlying molecular mechanisms remain unclear, and whether the attenuated inflammatory state is involved in ESC differentiation is completely unknown. Our recent study demonstrated that macroautophagy/autophagy-related protein ATG5 inhibits the inflammatory response of mouse ESCs (MmESCs) by promoting the degradation of BTRC/ß-TrCP1 and further the downregulation of NFKB/NF-κB signaling. In addition, maintenance of an attenuated inflammation status in MmESCs is required for their differentiation. In conclusion, ATG5 is a key regulator for the regulation of inflammatory response and differentiation of MmESCs.

Quercetin promotes ATG5-mediating autophagy-dependent ferroptosis in gastric cancer.

Quercetin has been documented to possess a multitude of pharmacological effects, encompassing antioxidant, antiviral, antimicrobial, and anti-inflammatory properties. Nevertheless, the exact molecular mechanisms responsible for the anti-tumor properties of quercetin remain to be fully explicated. To this end, quercetin was administered to gastric cancer cells (in vitro) AGS and MKN45, as well as BALB/c mice (in vivo). The proliferation ability of cells was evaluated using cholecystokinin octapeptide (CCK-8) and colony formation assays. The evaluation of ferroptosis involved the measurement of iron, malondialdehyde (MDA), and lipid reactive oxygen species. Autophagy and apoptosis were evaluated using immunofluorescence staining, western blotting, and flow cytometry analysis. Our findings indicate that quercetin significantly inhibited cell viability and tumor volume compared to the control group. Additionally, quercetin was found to decrease glutathione (GSH), malondialdehyde, and reactive oxygen species (ROS) levels while suppressing beclin1 and LC3B levels in cancer cells. Remarkably, the utilization of siATG5 was found to reverse all the aforementioned effects of quercetin. Ultimately, the effects of quercetin on gastric cancer were validated. In summary, our findings provide evidence that quercetin facilitates autophagy-mediated ferroptosis in gastric cancer.