Autophagy-related Markers Research Articles

High glucose levels delay the senescence of stem cells from human exfoliated deciduous teeth by suppressing autophagy

ObjectiveIn this study, we aimed to investigate the effects of varying glucose concentrations on the proliferation and senescence of stem cells from human exfoliated deciduous teeth (SHED) compared to human bone marrow-derived mesenchymal stem cells (hBMSC), and preliminarily clarify the difference of glucose metabolism between SHED and hBMSC. DesignWe cultured SHED and hBMSC in the presence of increasing glucose concentrations to study the role of glucose in cell viability, proliferation, and senescence. Gene expression related to the stemness of mesenchymal stem cells was evaluated using real-time quantitative reverse transcription-polymerase chain reaction. In addition, glucose consumption, lactic acid production, oxidative phosphorylation, and glycolysis were measured to analyze glucose metabolism and expression of autophagy-related markers, including microtubule-associated proteins 1 A/1B light chain 3 B and p62. ResultsWhile a high glucose level (4.5 g/L) promoted the proliferation of both SHED and hBMSC, it delayed senescence in SHED via autophagy inhibition but accelerated hBMSC senescence. In contrast to that in hBMSC, glycolysis in SHED was enhanced under the high-glucose culture condition. ConclusionsThe glycometabolism of SHED and hBMSC differed, and a high glucose culture medium was more favorable for SHED.

Saponins of Panax japonicus ameliorates cardiac aging phenotype in aging rats by enhancing basal autophagy through AMPK/mTOR/ULK1 pathway

Heart disease is a significant health concern for elderly individuals, with heart aging being the primary cause. Recent studies have shown that autophagy can play a protective role in preventing cardiac aging. Our previous research confirmed that Chikusetsu saponin IVa, a fundamental component of Saponins of Panax japonics (SPJ), can enhance basic autophagy levels in cardiomyocyte of isoproterenol induced cardiac fibrosis mice. However, it remains unclear whether SPJ possesses a protective effect on cardiac dysfunction during the natural aging process. Rats were randomly divided into four groups: adult control group (6 months old), aging group (24 months old), aging group treated with 10 mg/kg SPJ, and aging group treated with 30 mg/kg SPJ. The heart function, blood pressure, and heart mass index (HMI) were measured. Hematoxylin and eosin staining (H&E) and Wheat Germ Agglutinin (WGA) staining were used to observe the changes in morphology, while Masson staining was used to examine collagen deposition in the rat hearts and CD45 immunohistochemistry was conducted to examine the macrophage infiltration in heart tissues. TUNEL kit was used to detect apoptosis level of cardiomyocyte, and western blot was used to evaluate autophagy-related proteins as well as AMPK/mTOR/ULK1 pathway-related markers. SPJ treatment improved the cardiac function, reduced HMI, attenuated myocardial fiber disorder, inhibited inflammatory cell infiltration, and decreased collagen deposition and cardiomyocyte apoptosis in aging rats. Additionally, SPJ treatment decreased the expression of aging-related proteins and restored the expression of autophagy-related markers. SPJ activated autophagy through the activation of AMPK, which in turn increased the phosphorylation of ULK1(Ser555), while inhibited the phosphorylation of mTOR and ULK1(Ser757). Our study demonstrates that SPJ improves the cardiac function of aging rats by enhancing basal autophagy through the AMPK/mTOR/ULK1 pathway. These results offer a theoretical foundation and empirical evidence to support the clinical advancement of SPJ in enhancing age-related cardiac dysfunction.

Tanshinone IIA Regulates MAPK/mTOR Signal-Mediated Autophagy to Alleviate Atherosclerosis through the miR-214-3p/ATG16L1 Axis.

Tanshinone IIA (Tan IIA), the core ingredient of Salvia miltiorrhiza, is commonly used for treating cardiovascular diseases. However, its underlying mechanism in regulating autophagy in atherosclerosis (AS) remains unclear. An in vivo model of AS was constructed using Apolipoprotein E-deficient (ApoE-/-) mice fed with a high-fat diet. Histopathologic changes and lipid accumulation were evaluated by hematoxylin and eosin (HE) and Oil red O staining, respectively. The inflammatory cytokine levels were evaluated by Enzyme-linked immunosorbent assay (ELISA). An oxidized low-density lipoprotein (ox-LDL) was used to induce foam cells in RAW264.7 cells. Cholesterol uptake and efflux assay were used to assess changes in intracellular and extracellular cholesterol levels. The expression levels of autophagy-related protein-16-like protein 1 (ATG16L1) and miR-214-3p in the samples and cells derived from mice were assessed by quantitative real-time polymerase chain reaction (qRT-PCR), and the protein levels of the mitogen-activated protein kinase (MAPK)/mammalian target of rapamycin (mTOR) and autophagy-related markers were detected using western blot. The binding site of miR-214-3p on ATG16L1 was determined using a dual-luciferase reporter assay. We observed a decrease in ATG16L1 and increase in miR-214-3p expression level in the AS mice and ox-LDL stimulated RAW264.7 cells. However, the miR-214-3p and ATG16L1 expression could be reversed by Tan IIA. In vivo experiments showed that Tan IIA alleviated AS by reducing lipid accumulation and inflammatory factor levels and promoting autophagy. The in vitro assays demonstrated that Tan IIA regulated lipid levels and autophagy via the miR-214-3p/ATG16L1 axis to inhibit foam cell formation. Additionally, Tan IIA inhibited the MAPK/mTOR pathway by reducing miR-214-3p expression and promoting autophagy. Findings from this study suggested that Tan IIA regulated the MAPK/mTOR signal-mediated autophagy to alleviate AS through the miR-214-3p/ATG16L1 axis.

Macrophage migration inhibitory factor exacerbates asthmatic airway remodeling via dynamin-related protein 1-mediated autophagy activation

BackgroundMacrophage migration inhibitory factor (MIF) and GTPase dynamin-related protein 1 (Drp1)-dependent aberrant mitochondrial fission are closely linked to the pathogenesis of asthma. However, it is unclear whether Drp1-mediated mitochondrial fission and its downstream targets mediate MIF-induced proliferation of airway smooth muscle cells (ASMCs) in vitro and airway remodeling in chronic asthma models. The present study aims to clarify these issues.MethodsIn this study, primary cultured ASMCs and ovalbumin (OVA)-induced asthmatic rats were applied. Cell proliferation was detected by CCK-8 and EdU assays. Western blotting was used to detect extracellular signal-regulated kinase (ERK) 1/2, Drp1, autophagy-related markers and E-cadherin protein phosphorylation and expression. Inflammatory cytokines production, airway reactivity test, histological staining and immunohistochemical staining were conducted to evaluate the development of asthma. Transmission electron microscopy was used to observe the mitochondrial ultrastructure.ResultsIn primary cultured ASMCs, MIF increased the phosphorylation level of Drp1 at the Ser616 site through activation of the ERK1/2 signaling pathway, which further activated autophagy and reduced E-cadherin expression, ultimately leading to ASMCs proliferation. In OVA-induced asthmatic rats, MIF inhibitor 4-iodo-6-phenylpyrimidine (4-IPP) treatment, suppression of mitochondrial fission by Mdivi-1 or inhibiting autophagy with chloroquine phosphate (CQ) all attenuated the development of airway remodeling.ConclusionsThe present study provides novel insights that MIF promotes airway remodeling in asthma by activating autophagy and degradation of E-cadherin via ERK/Drp1 signaling pathway, suggesting that targeting MIF/ERK/Drp1 might have potential therapeutic value for the prevention and treatment of asthma.

Metformin protects human lens epithelial cells from high glucose-induced senescence and autophagy inhibition by upregulating SIRT1.

The aim of this study is to explore whether metformin (MET) protects the human lens epithelial cells (HLECs) from high glucose-induced senescence and to identify the underlying mechanisms. A cellular senescence model was established by treating HLE-B3 cells with D-glucose and then intervened with MET. Concentrations of high glucose (HG) and MET were detected using CCK-8 and western blot. qRT-PCR, western blot, and senescence-associated β-galactosidase (SA-β-gal) were performed to verify the protective effect of MET on senescent HLE-B3 cells. Additionally, western blot and qRT-PCR were conducted to detect the effects of MET on autophagy-related markers p62 and LC3, as well as SIRT1. In vitro, we observed apparent senescence in human lens epithelial cells (HLECs) under high glucose conditions. This was characterized by increased senescence-associated genes p21 and p53. However, the addition of MET significantly reduced the occurrence of HLECs senescence. We also observed that high glucose inhibited both autophagy and SIRT1, which could be restored by MET. Moreover, we verified that the anti-senescence effect of MET was mediated by SIRT1 using SIRT1 activators and inhibitors. We have demonstrated that autophagy and SIRT1 activity are inhibited in HLE-B3 cells using the HG induced senescence model. Furthermore, our results showed that MET can delay senescence by activating SIRT1 and autophagy. These findings suggest that MET may be a promising candidate for alleviating cataract development and provide a direction for further investigation into the underlying molecular mechanisms.

Aucubin alleviates methotrexate-induced enteritis in rats by inducing autophagy.

One of the toxic side effects of methotrexate (MTX) is enteritis. Aucubin, an iridoid glycoside derived from traditional medicinal herbs, has been proven to have anti-inflammation, anti-apoptosis and anti-oxidation properties. This work explored the effect and mechanism of aucubin in treating MTX-induced enteritis in a rat model. Two doses of aucubin (5 and 10 mg/kg) were adopted for the assessment of its pharmacological activity. We observed that in rats with MTX-induced enteritis, the body weight and small intestinal weight decreased. The intestine barrier was injured, as reflected by pathological examinations and an increase in D-lactate and diamine oxidase concentration in serum. Intestinal inflammation was shown by the observation of macrophages in the intestine and the concentrations of inflammatory cytokines tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in serum. The NLR family pyrin domain containing 3 (NLRP3) inflammasome was shown to be activated by the enhancement of NLRP3, cleaved-caspase 1, IL-18 and IL-1β. Moreover, autophagy was reflected by transmission electron microscopy as slightly induced, along with changes in autophagy-related markers microtubule-associated protein 1 light chain 3 (LC3) and Beclin1. Remarkably, aucubin treatment attenuated the MTX-induced disease activity index increase, intestinal damage, inflammatory response and NLRP3 inflammasome activation, but provoked autophagy. Rapamycin, an autophagy activator, showed similar therapeutic effects to aucubin on MTX-induced enteritis. However, 3-methyladenine, an autophagy inhibitor, reversed the protective effects of aucubin. These findings prompted the hypothesis that aucubin alleviates MTX-induced enteritis by aggravating autophagy. This study might provide evidence for further investigation on the therapeutic role of aucubin in MTX-resulted enteritis.

Enhanced Autophagy in Damaged Laminar Tissue of Acute Laminitis Induced by Oligofructose Overloading in Dairy Cows.

This study was aimed at determining the autophagy activity in the laminar tissue of dairy cows with oligofructose-induced laminitis. Twelve healthy non-pregnant Holstein cows were randomly divided into two groups of six cows each, entitled the control group and the oligofructose overload group (OF group), respectively. At 0 h, cows in the OF group were gavaged with oligofructose (17 g/kg BW) dissolved in warm deionized water (20 mL/kg BW) through an oral rumen tube, and the dairy cows in the control group were gavaged with the same volume of deionized water by the same method. At -72 h before, as well as 0 h, 6 h, 12 h, 18 h, 24 h, 36 h, 48 h, 60 h, and 72 h after perfusion, clinical evaluations of both groups were monitored. After 72 h, the laminar tissues of the dairy cows in both groups were collected to examine the genes and proteins. The gene expression of ATG5, ATG12, and Beclin1 significantly increased (p < 0.05), whereas that of P62 and mTOR significantly decreased (p < 0.01) in the OF group relative to the control group. The protein expression of Beclin-1 significantly increased (p < 0.05), while that of LC3II significantly decreased (p < 0.05) in the OF group relative to the control group. However, the protein expression of P62 non-significantly reduced (p > 0.05) in the OF group comparative to the control group. Furthermore, the distribution of the Beclin1 protein in the laminar tissue significantly increased (p < 0.01), while that of the P62 protein significantly decreased (p < 0.05) in the OF group than the control group. These findings indicate that the imbalanced gene and protein-level status of autophagy-related markers may be the basic cause for the failure of the epidermal attachment. However, a more detailed gene and protein-level study is needed to further clarify the role of autophagy in the pathogenesis of bovine laminitis.

The miR-224-5p/SIRT3/AMPK/mTOR axis is involved in the melatonin-mediated inhibition of glucocorticoid-induced osteoporosis by activating autophagy.

Melatonin has been shown to exert an inhibitory effect on osteoporosis. This study investigates the function of the miR-224-5p/SIRT3/AMPK/mTOR axis in melatonin-mediated effects against osteoporosis. Human bone marrow mesenchymal stem cells (hBMSCs) were treated with glucocorticoid dexamethasone to induce an in vitro osteoporosis model. After melatonin treatment, miR-224-5p and SIRT3 levels were measured by RT‒PCR. Transmission electron microscopy and immunofluorescence were conducted for evaluating autophagy. Western blotting was carried out to determine the expression of osteogenesis-related proteins (Runx2, OSX, OPN, and OCN), SIRT3-AMPK-mTOR axis, and autophagy-related markers (LC3 and p62). Alizarin red staining was used to measure matrix mineralization. The data showed that melatonin inhibited dexamethasone-induced osteoporosis in vitro, and enhanced autophagic levels (as indicated by increased LC3 puncta, LC3II/I ratio, and autophagic vacuoles). In terms of the mechanisms, melatonin decreased miR-224-5p expression and increased SIRT3. SRIT3 was shown to be a direct target of miR-224-5p. miR-224-5p upregulation or SIRT3 downregulation reversed the effects of melatonin on osteoporosis and suppressed autophagy. Additionally, miR-224-5p inhibited SIRT3 expression and AMPK pathway activation. In summary, we discovered that melatonin suppressed glucocorticoid-induced osteoporosis and autophagy inhibition via the miR-224-5p/SIRT3/AMPK/mTOR axis.

Increased apoptosis of gingival epithelium is associated with impaired autophagic flux in medication-related osteonecrosis of the jaw

ABSTRACT Macroautophagy/autophagy has both negative and positive aspects in the development of many diseases. Yet, its exact role and specific mechanism in the onset of medication-related osteonecrosis of the jaw (MRONJ) is still not fully understood. Retarded gingiva healing is the primary clinical manifestation in patients with MRONJ. In this study, we aimed to explore the relationship between autophagy and apoptosis in MRONJ gingival epithelium and search for a method to prevent this disease. First, we examined clinical samples from patients diagnosed with MRONJ and healthy controls, finding that autophagy-related markers MAP1LC3/LC3 and SQSTM1/p62 synchronously increased, thus suggesting that autophagic flux was suppressed in MRONJ. Moreover, mRNA sequencing analysis and TUNEL assay showed that the process of apoptosis was upregulated in patients and animals with MRONJ, indicating autophagy and apoptosis participate in the development of MRONJ. Furthermore, the level of autophagy and apoptosis in zoledronic acid (ZA)-treated human keratinocytes cell lines (HaCaT cells) was concentration dependent in vitro. In addition, we also found that RAB7 (RAB7, member RAS oncogene family) activator ML098 could rescue MRONJ gingival lesions in mice by activating the autophagic flux and downregulating apoptosis. To sum up, this study demonstrated that autophagic flux is impaired in the gingival epithelium during MRONJ, and the rescued autophagic flux could prevent the occurrence of MRONJ. Abbreviations: ACTB: actin beta; Baf-A1: bafilomycin A1; CASP3: caspase 3; CASP8: caspase 8; CT: computed tomography; DMSO: dimethyl sulfoxide; GFP: green fluorescent protein; HaCaT cells: human keratinocytes cell lines; H&E: hematoxylin and eosin; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MRONJ: medication-related osteonecrosis of the jaw; PARP: poly(ADP-ribose) polymerase; RAB7: RAB7, member RAS oncogene family; RFP: red fluorescent protein; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; ZA: zoledronic acid.

Exogenous galanin alleviates hepatic steatosis by promoting autophagy via the AMPK-mTOR pathway

Defective autophagy-induced intracellular lipid degradation is causally associated with non-alcoholic fatty liver disease (NAFLD) development. Therefore, agents that can restore autophagy may have potential clinical application prospects on this public health issue. Galanin (GAL) is a pleiotropic peptide that regulates autophagy and is a potential drug for the treatment of NAFLD. In this study, we used an MCD-induced NAFLD mouse model in vivo and an FFA-induced HepG2 hepatocyte model in vitro to evaluate the anti-NAFLD effect of GAL. Exogenous GAL supplementation significantly attenuated lipid droplet accumulation and suppressed hepatocyte TG levels in mice and cell models. Mechanistically, Galanin-mediated reduction of lipid accumulation was positively correlated with upregulated p-AMPK, as evidenced by upregulated protein expressions of fatty acid oxidation-related gene markers (PPAR-α and CPT1A), upregulated expressions of the autophagy-related marker (LC3B), and downregulated autophagic substrate p62 levels. In FFA-treated HepG2 cells, activation of fatty acid oxidation and autophagy-related proteins by galanin was reversed by autophagy inhibitors, chloroquine, and the AMPK inhibitor. Galanin ameliorates hepatic fat accumulation by inducing autophagy and fatty acid oxidation via the AMPK/mTOR pathway.

Fluoride induces osteoblast autophagy by inhibiting the PI3K/AKT/mTOR signaling pathway in vivo and in vitro.

Fluorosis primarily manifests as bone damage in the form of dental fluorosis and skeletal fluorosis and represents a critical global public health challenge. However, few studies have examined autophagy-related signaling pathways in skeletal fluorosis. This study aimed to investigate the effect of fluoride on autophagy in osteoblasts using comprehensive methods and to explore the role of the PI3K/AKT/mTOR signaling pathway in regulating fluoride-induced autophagy in osteoblasts. Sprague-Dawley (SD) rats were exposed to different concentrations of fluoride (NaF: 5, 50, and 100 mg/L) for six months. Primary osteoblasts were treated with 0.5, 1.0, or 3.0 mM NaF. Hematoxylin and eosin (H&E) staining, transmission electron microscopy (TEM), immunohistochemistry (IHC), immunofluorescence staining, and western blotting were performed to evaluate morphological changes in bone tissues and autophagosomes and to detect the protein expression of autophagy-related markers and PI3K/AKT/mTOR signaling pathway-related molecules both in vivo and in vitro. The bone tissues of fluoride-exposed rats showed osteosclerosis, autophagosomes and autolysosomes. LC3B immunofluorescence staining revealed an increase in autophagosomes in the primary osteoblasts treated with fluoride. The LC3Ⅱ/Ⅰ ratio and levels of autophagy-related markers (Beclin 1 and Atg7) were increased, whereas P62 levels were decreased in bone tissues and primary osteoblasts in the fluoride groups. Simultaneously, p-AKT and p-mTOR levels were reduced in bone tissues and primary osteoblasts in the fluoride groups. Moreover, a PI3K inhibitor (LY294002) further downregulated p-AKT and p-mTOR protein expression but slightly increased the LC3Ⅱ/Ⅰ ratio in primary osteoblasts. These results demonstrate that fluoride induces autophagy in osteoblasts by inhibiting the PI3K/AKT/mTOR signaling pathway, which deepens our understanding of the molecular mechanisms underlying fluoride-induced bone damage and provides a theoretical basis for the prevention and treatment of skeletal fluorosis.

HIV Tat-mediated altered oligodendrocyte maturation involves autophagy-lysosomal dysfunction

Abstract Objectives The current study was undertaken to understand the underlying molecular mechanism(s) by which HIV Transactivator of transcription (Tat) alters oligodendrocyte maturation through the generation of reactive oxygen species (ROS), impairment of lysosomal functioning, and dysregulation of autophagy. Methods We exposed primary rat immature oligodendrocytes to HIV Tat and utilized various experimental techniques to assess its effects on oligodendrocytes maturation. We measured ROS levels, assessed lysosomal membrane potential, determined cathepsin D activity, and analyzed the expression of autophagy-related markers. Furthermore, we investigated the potential of ROS scavengers and lysosomal protectants to mitigate the damaging effects of HIV Tat on oligodendrocytes maturation. Results Exposure of primary rat immature oligodendrocytes to HIV Tat significantly increased ROS levels, indicating the induction of oxidative stress. This oxidative stress impaired lysosomal functioning, as evidenced by a substantial increase in lysosomal membrane potential and a decrease in cathepsin D activity. Compromised lysosomal function resulted in dysregulated autophagy, which was confirmed by increased expression of SQSTM1. However, the administration of ROS scavengers and lysosomal protectants effectively attenuated the detrimental effects of HIV Tat on oligodendrocytes maturation. Conclusions Our findings demonstrate that HIV Tat exposure induces oxidative stress, impairs lysosomal functioning, and dysregulates autophagy in oligodendrocytes. These molecular changes likely contribute to the altered maturation of oligodendrocytes observed in HIV-infected individuals. Understanding these underlying mechanisms provides valuable insights into the pathogenesis of HIV-associated neurocognitive disorders and highlights the potential of therapeutic strategies targeting ROS scavenging and lysosomal protection as adjunctive approaches for managing such complications in HIV +ve individuals.

Identification and cross-validation of autophagy-related genes in cardioembolic stroke.

Cardioembolic stroke (CE stroke, also known as cardiogenic cerebral embolism, CCE) has the highest recurrence rate and fatality rate among all subtypes of ischemic stroke, the pathogenesis of which was unclear. Autophagy plays an essential role in the development of CE stroke. We aim to identify the potential autophagy-related molecular markers of CE stroke and uncover the potential therapeutic targets through bioinformatics analysis. The mRNA expression profile dataset GSE58294 was obtained from the GEO database. The potential autophagy-related differentially expressed (DE) genes of CE stroke were screened by R software. Protein-protein interactions (PPIs), correlation analysis, and gene ontology (GO) enrichment analysis were applied to the autophagy-related DE genes. GSE66724, GSE41177, and GSE22255 were introduced for the verification of the autophagy-related DE genes in CE stroke, and the differences in values were re-calculated by Student's t-test. A total of 41 autophagy-related DE genes (37 upregulated genes and four downregulated genes) were identified between 23 cardioembolic stroke patients (≤3 h, prior to treatment) and 23 healthy controls. The KEGG and GO enrichment analysis of autophagy-related DE genes indicated several enriched terms related to autophagy, apoptosis, and ER stress. The PPI results demonstrated the interactions between these autophagy-related genes. Moreover, several hub genes, especially for CE stroke, were identified and re-calculated by Student's t-test. We identified 41 potential autophagy-related genes associated with CE stroke through bioinformatics analysis. SERPINA1, WDFY3, ERN1, RHEB, and BCL2L1 were identified as the most significant DE genes that may affect the development of CE stroke by regulating autophagy. CXCR4 was identified as a hub gene of all types of strokes. ARNT, MAPK1, ATG12, ATG16L2, ATG2B, and BECN1 were identified as particular hub genes for CE stroke. These results may provide insight into the role of autophagy in CE stroke and contribute to the discovery of potential therapeutic targets for CE stroke treatment.

SIRT3-mediated autophagy contributes to ferroptosis-induced anticancer by inducing the formation of BECN1-SLC7A11 complex

Ferroptosis is an autophagy-dependent cell death associated with iron accumulation and lipid peroxidation, which plays a crucial part in anticancer activity. Sirtuin 3 (SIRT3) positively regulates autophagy by phosphorylation of activated protein kinase (AMPK). However, whether SIRT3-mediated autophagy can inhibit the cystine/glutamate antiporter (system Xc−) activity by inducing the formation of a BECN1-SLC7A11 complex and consequently promote induction of ferroptosis is unknown. Using both in vitro and in vivo models, we revealed that combination treatment with erastin and TGF-β1 decreased the expression of epithelial-mesenchymal transition-related markers and inhibited the invasion and metastasis of breast cancer. Furthermore, TGF-β1 promoted erastin-induced ferroptosis-related indicators in MCF-7 cells and tumor-bearing nude mice models. Interestingly, the expression of SIRT3, p-AMPK, and autophagy-related markers were significantly elevated after co-treatment with erastin and TGF-β1, suggesting that combination treatment of erastin and TGF-β1 mediated autophagy by the SIRT3/AMPK signaling pathway. In addition, erastin-induced BECN1-SLC7A11 complexes were more abundant after co-treatment with TGF-β1. This effect was inhibited by the autophagy inhibitor 3-methyladenine or siSIRT3, further revealing that combination treatment of erastin and TGF-β1 mediated autophagy-dependent ferroptosis by inducing the formation of BECN1-SLC7A11 complexes. Our results agreed with the concept that BECN1 directly binds to SLC7A11 to inhibit system Xc− activity. In summary, our studies confirmed that SIRT3-mediated autophagy is conducive to ferroptosis-mediated anticancer activity by inducing the formation of BECN1-SLC7A11 complexes, which is a potential therapeutic approach for treating breast cancer.

Esketamine is neuroprotective against traumatic brain injury through its modulation of autophagy and oxidative stress via AMPK/mTOR-dependent TFEB nuclear translocation

Recent clinical studies highlight the neuroprotective effects of esketamine, but its benefits following traumatic brain injury (TBI) have not been defined. Here, we investigated the effects of esketamine following TBI and its associated neuroprotection mechanisms. In our study, controlled cortical impact injury on mice was utilized to induce the TBI model in vivo. TBI mice were randomized to receive vehicle or esketamine at 2 h post-injury for 7 consecutive days. Neurological deficits and brain water content in mice were detected, respectively. Cortical tissues surrounding focal trauma were obtained for Nissl staining, immunofluorescence, immunohistochemistry, and ELISA assay. In vitro, esketamine were added in culture medium after cortical neuronal cells induced by H2O2 (100μM). After exposed for 12h, neuronal cells were obtained for western blotting, immunofluorescence, ELISA and CO-IP assay. Following administration of 2–8 mg/kg esketamine, we observed that 8 mg/kg esketamine produced no additional recovery of neurological function and ability to alleviate brain edema in TBI mice model, so 4 mg/kg esketamine was selected for subsequent experiments. Additionally, esketamine can effectively reduce TBI-induced oxidative stress, the number of damaged neurons, and the number of TUNEL-positive cells in the cortex of TBI models. Meanwhile, the levels of Beclin 1, LC3 II, and the number of LC3-positive cells in injured cortex were also increased following esketamine exposure. Western blotting and immunofluorescence assays showed that esketamine accelerated the nuclear translocation of TFEB, increased the p-AMPKα level and decreased the p-mTOR level. Similar results including nuclear translocation of TFEB, the increases of autophagy-related markers, and influences of AMPK/mTOR pathway were observed in H2O2-induced cortical neuronal cells; however, BML-275 (AMPK inhibitor) can reverse these effects of esketamine. Furthermore, TFEB silencing not only decreased the Nrf2 level in H2O2-induced cortical neuronal cells, but also alleviated the oxidative stress. Importantly, CO-IP confirmed the interaction between TFEB and Nrf2 in cortical neuronal cells. These findings suggested that esketamine exerts the neuroprotective effects of esketamine in TBI mice model via enhancing autophagy and alleviating oxidative stress; its mechanism involves AMPK/mTOR-dependent TFEB nuclear translocation-induced autophagy and TFEB/Nrf2-induced antioxidant system.

Yi-Qi-Jian-Pi formula ameliorates immune function in acute-on-chronic liver failure by upregulating autophagy and mitochondrial biogenesis in CD8+ T lymphocytes.

A key event in the pathogenesis of acute-on-chronic liver failure (ACLF) is the imbalance in the systemic immune response; immunosuppression in patients with ACLF contributes to poor prognosis. The Yi-Qi-Jian-Pi formula (YQJPF) may improve T lymphocyte immune function in patients with ACLF. To investigate the immune mechanism of YQJPF in vivo and in vitro. An ACLF rat model was established by injection of CCl4, lipopolysaccharide, and D-galactosamine. We examined the effect of different doses of YQJPF (6.43, 12.87, 25.74g/kg) on liver injury and immune function in the ACLF rat model. Magnetic-activated cell sorting was used to sort the CD8+ T lymphocytes in the spleen for lymphocyte function detection. In primary CD8+ T lymphocytes and Jurkat cell lines, the expression of mitochondrial function and biogenesis and autophagy related markers were detected using molecular biological methods and flow cytometry analysis. YQJPF improved the peripheral blood lymphocyte count and proportion of CD8+ T lymphocytes in ACLF rats, increased pro-inflammatory factors (IL-2, IFN-λ, and TNF-α), and reduced anti-inflammatory factors (IL-10 and TGF β1). YQJPF also improved metabolism and mitochondrial homeostasis in CD8+ T lymphocytes, alleviated lymphocyte immune dysfunction by promoting autophagy, upregulated mitochondrial biogenesis by promoting PGC-1α, NRF-1, and TFAM expression, and regulated the relationship between autophagy and mitochondrial biogenesis via PGC-1α. Our results suggest that YQJPF could improve immune function in a rat model of ACLF, possibly via affecting the homeostasis of lymphatic mitochondria in CD8+ T lymphocytes. YQJPF may enhance lymphocyte mitochondrial biosynthesis and promote lymphocyte autophagy. PGC-1α is a possible upstream regulatory target of YQJPF.

LncRNA GAS5 regulated by FTO-mediated m6A demethylation promotes autophagic cell death in NSCLC by targeting UPF1/BRD4 axis.

Long non-coding RNA (lncRNA) growth arrest-specific transcript 5 (GAS5) has been shown to be a regulator for many cancers, including non-small cell lung cancer (NSCLC). Therefore, its role and mechanism in the process of NSCLC deserve to be further revealed. The expression levels of GAS5, fat mass and obesity-associated protein (FTO) and bromodomain-containing protein 4 (BRD4) were detected by quantitative real-time PCR. Western blot analysis was used to examine the protein expression of FTO, BRD4, up-frameshift protein 1 (UPF1) and autophagy-related markers. Methylated RNA immunoprecipitation was used to assess the m6A level of GAS5 regulated by FTO. Cell proliferation and apoptosis were determined using MTT assay, EdU assay and flow cytometry. Autophagy ability was assessed by immunofluorescence staining and transmission electron microscope. Xenograft tumor model was constructed to explore the effects of FTO and GAS5 on NSCLC tumor growth in vivo. The interaction between UPF1 and GAS5 or BRD4 was confirmed by pull-down assay, RIP assay, dual-luciferase reporter assay, and chromatin immunoprecipitation. Fluorescent in situ hybridization was used to analyze the co-localization of GAS5 and UPF1. Actinomycin D treatment was employed to evaluate BRD4 mRNA stability. GAS5 was downregulated in NSCLC tissues and was associated with poor prognosis in NSCLC patients. FTO was highly expressed in NSCLC, and it inhibited GAS5 expression by reducing GAS5 m6A methylation level. GAS5 suppressed by FTO could promote the autophagic death of NSCLC cells in vitro and inhibit NSCLC tumor growth in vivo. In addition, GAS5 was able to interact with UPF1 to reduce the mRNA stability of BRD4. Knockdown of BRD4 reversed the inhibition of GAS5 or UPF1 silencing on the autophagic cell death of NSCLC. The findings of the study showed that lncRNA GAS5 mediated by FTO could contribute to the autophagic cell death of NSCLC by interacting with UPF1 to reduce BRD4 mRNA stability, suggesting that GAS5 might be a vital therapy target for NSCLC progression.

A novel signature of autophagy-related immunophenotyping biomarkers in osteoarthritis

AimsWe aimed to provide an autophagy-related signature to seek immunophenotyping biomarkers in osteoarthritis (OA). Materials and methodsMicroarray expression profiling of OA subchondral bone samples and screening of an autophagy database for autophagy-related differentially expressed genes (au-DEGs) between OA and normal samples were performed. A weighted gene co-expression network analysis (WGCNA) was constructed using au-DEGs to identify key modules significantly associated with clinical information of OA samples. OA-related autophagy hub genes were identified based on the connectivity with the phenotypes of genes in key modules and the protein-protein interaction (PPI) network in which the genes in the modules are involved, followed by feasibility verification of autophagy hub genes by bioinformatics analysis and biological experiments. Key findingsWe screened 754 au-DEGs between OA and control samples, and co-expression networks were constructed using au-DEGs. Three OA-related autophagy hub genes (HSPA5, HSP90AA1, and ITPKB) were identified. Based on the hub gene expression profiles, OA samples were divided into two clusters with significantly different expression profiles and distinct immunological features, and the three hub genes were significantly differentially expressed between the clusters. Differences in hub genes between OA and control samples regarding sex, age, and grades of OA were examined using external datasets and experimental validation. SignificanceThree autophagy-related markers of OA were identified using bioinformatics methods, and these markers may be useful for the autophagy-related immunophenotyping of OA. The present data may facilitate the diagnosis of OA, as well as the design of immunotherapies and individualized medical treatments.

Dexmedetomidine improves acute lung injury by activating autophagy in a rat hemorrhagic shock and resuscitation model

Dexmedetomidine (DEX) can reduce lung injury in a hemorrhagic shock (HS) resuscitation (HSR) model in rats by inhibiting inflammation. Here, we aimed to investigate if these effects of DEX are due to autophagy activation. Therefore, we established HSR rat models and divided them into four groups. HS was induced using a blood draw. The rats were then resuscitated by reinjecting the drawn blood and saline. The rats were sacrificed 24 h after resuscitation. Lung tissues were harvested for histopathological examination, determination of wet/dry lung weight ratio, and detection of the levels of autophagy-related marker proteins LC3, P62, Beclin-1, and the ATG12-ATG5 conjugate. The morphological findings of hematoxylin and eosin staining in lung tissues and the pulmonary wet/dry weight ratio showed that lung injury improved in HSR + DEX rats. However, chloroquine (CQ), an autophagy inhibitor, abolished this effect. Detecting the concentration of autophagy-related proteins showed that DEX administration increased LC3, ATG12-ATG5, and Beclin-1 expression and decreased P62 expression. The expression levels of these proteins were similar to those in the HSR group after CQ + DEX administration. In summary, DEX induced autophagic activation in an HSR model. These findings suggest that DEX administration partially ameliorates HSR-induced lung injury via autophagic activation.

Hederacolchiside A1 Suppresses Autophagy by Inhibiting Cathepsin C and Reduces the Growth of Colon Cancer.

While autophagy degrades non-functional or unnecessary cellular components, producing materials for synthesizing cellular components, it can also provide energy for tumor development. Hederacolchiside A1 (HA1) derived from anemone raddeana has anticancer effects on several carcinomas by inducing apoptosis or exhibiting cytotoxicity, but the relationship with autophagy has not been studied. We investigated the association between HA1 and autophagy and evaluated its anticancer effect on colon cancer. HA1 induced accumulation of the autophagy-related markers LC3B and SQSTM1, with distinct vacuolar formation, unlike other autophagy inhibitors; the effects were similar to those of chloroquine. In addition, HA1 decreased the expression and proteolytic activity of lysosomal protein cathepsin C, reduced the growth of colon cancer cells in vitro, and inhibited tumor growth in vivo. It also reduced the expression of Ki-67 and cathepsin C in mouse tissues and reduced the growth of spheroids and organoids composed of cancer cells. Taken together, these results imply that HA1 regulates cell growth and autophagy and has potential as a promising therapeutic agent in colon cancer.