Effect of tankyrase 1 on autophagy in the corpus cavernosum smooth muscle cells from ageing rats with erectile dysfunction and its potential mechanism

This study compared tankyrase 1 expression and autophagy quantity between erectile dysfunction （ED） and non-ED rats＇ corpus cavernosum smooth muscle cells （CSMCs）. This study aslo explored the effect and possible mechanism of tankyrase 1 on autophagy and cell proliferation in ageing ED rats＇ CSMCs. The intracavernous pres- sure and mean systemic arterial pressure were measured to investigate erectile function so that eight 24-month-old ED and eight 8-month-old male Wistar rats were choosed respectively. The rat CSMCs were isolated and cultured by enzyme digestion, in which tankyrase 1 expression and autophagy quantity were compared. Tankyrase 1 over-expression was induced with plasmid transfection by Lipofectamine^TM. The effect of tankyrase 1 overexpression on proliferation, autophagy and mTOR pathway in 24-month-old ED rats＇ CSMCs was measured by the cell growth curve in MTT assay, cell cycle analysis in flow cytometry （FCM）, key protein expression in Western blot, autophagy quantity in transmission electron microscopy, monodansylcadaverine staining and GFP-LC3 fluorescence. The primary CSMCs were confirmed by immunofluorescence, and the purity was 99.1% in FCM. Compared with that of 8-month-old rats, tankyrase 1 expression and autophagy quantity significantly decreased in 24-month-old ED rats＇ primary CSMCs （P 〈 0.01）. Tankyrase 1 overexpression significantly increased the growth rate （P 〈 0.05） and increased the S phase of cell cycle （P 〈 0.01）. The autophagosome quantity was remarkably increased （P 〈 0.01）, LC3-Ⅰ/Ⅱ and Beclin 1 were upregulated （P 〈 0.01 and P 〈 0.05）, and p-p70S6K （Thr389） was downregulated in 24-month-old ED rat CSMCs （P 〈 0.05）. In conclusion, Tankyrase 1 and autophagy decrease in the CSMCs from aging rats with ED, and tankyrase 1 may have a positive effect on proliferation by enhancing autophagy and regulating the mTOR signalling pathway.

Humanβ-defensin-1 affects the mammalian target of rapamycin pathway and autophagy in colon cancer cells through long noncoding RNA TCONS_00014506

BACKGROUND Colorectal cancer has a low 5-year survival rate and high mortality.Humanβ-defensin-1(hBD-1)may play an integral function in the innate immune system,contributing to the recognition and destruction of cancer cells.Long non-coding RNAs(lncRNAs)are involved in the process of cell differentiation and growth.AIM To investigate the effect of hBD-1 on the mammalian target of rapamycin(mTOR)pathway and autophagy in human colon cancer SW620 cells.METHODS CCK8 assay was utilized for the detection of cell proliferation and determination of the optimal drug concentration.Colony formation assay was employed to assess the effect of hBD-1 on SW620 cell proliferation.Bioinformatics was used to screen potentially biologically significant lncRNAs related to the mTOR pathway.Additionally,p-mTOR(Ser2448),Beclin1,and LC3II/I expression levels in SW620 cells were assessed through Western blot analysis.RESULTS hBD-1 inhibited the proliferative ability of SW620 cells,as evidenced by the reduction in the colony formation capacity of SW620 cells upon exposure to hBD-1.hBD-1 decreased the expression of p-mTOR(Ser2448)protein and increased the expression of Beclin1 and LC3II/I protein.Furthermore,bioinformatics analysis identified seven lncRNAs(2 upregulated and 5 downregulated)related to the mTOR pathway.The lncRNA TCONS_00014506 was ultimately selected.Following the inhibition of the lncRNA TCONS_00014506,exposure to hBD-1 inhibited p-mTOR(Ser2448)and promoted Beclin1 and LC3II/I protein expression.CONCLUSION hBD-1 inhibits the mTOR pathway and promotes autophagy by upregulating the expression of the lncRNA TCONS_00014506 in SW620 cells.

TAX1BP1 and FIP200 orchestrate non-canonical autophagy of p62 aggregates for mouse neural stem cell maintenance

Autophagy plays a pivotal role in diverse biological processes,including the maintenance and differentiation of neural stem cells(NSCs).Interestingly,while complete deletion of Fip200 severely impairs NSC maintenance and differentiation,inhibiting canonical autophagy via deletion of core genes,such as Atg5,Atg16l1,and Atg7,or blockade of canonical interactions between FIP200 and ATG13(designated as FIP200-4A mutant or FIP200 KI)does not produce comparable detrimental effects.This highlights the likely critical involvement of the non-canonical functions of FIP200,the mechanisms of which have remained elusive.Here,utilizing genetic mouse models,we demonstrated that FIP200 mediates non-canonical autophagic degradation of p62/sequestome1,primarily via TAX1BP1 in NSCs.Conditional deletion of Tax1bp1 in fip200hGFAP conditional knock-in(cKI)mice led to NSC deficiency,resembling the fip200hGFAP conditional knockout(cKO)mouse phenotype.Notably,reintroducing wild-type TAX1BP1 not only restored the maintenance of NSCs derived from tax1bp1-knockout fip200hGFAP cKI mice but also led to a marked reduction in p62 aggregate accumulation.Conversely,a TAX1BP1 mutant incapable of binding to FIP200 or NBR1/p62 failed to achieve this restoration.Furthermore,conditional deletion of Tax1bp1 in fip200hGFAP cKO mice exacerbated NSC deficiency and p62 aggregate accumulation compared to fip200hGFAP cKO mice.Collectively,these findings illustrate the essential role of the FIP200-TAX1BP1 axis in mediating the non-canonical autophagic degradation of p62 aggregates towards NSC maintenance and function,presenting novel therapeutic targets for neurodegenerative diseases.

CircR-ZC3HC1 mediates MiR-384-5p/SIRT1 axis to promote neuronal autophagy and relieves ischemic stroke

Objective:Circular RNAs(circRNAs)have been shown to involve in pathological processes of ischemic stroke(IS),including autophagy.This study was designed to explore the effect of circR-ZC3HC1 on neuronal autophagy in IS and the related mechanisms.Methods:Expression of circR-ZC3HC1 in blood samples of IS patients and healthy controls was detected.Hippocampal neurons were treated with oxygen and glucose deprivation(OGD)to establish IS in vitro model.The expression of LC3 and p62 and the number of autophagosomes were examined to evaluate the autophagy level of OGD induced neurons using western blotting and transmission electron microscope.Cell apoptosis rate and the expression of cleaved caspase-3,Bax,and Bcl-2 were assessed byflow cytometry and western blotting.The binding relationships among circR-ZC3HC1,miR-384-5p,and SIRT1 were predicted and verified.Results:Low expression of circR-ZC3HC1 was found in blood samples of IS patients and OGD-treated neurons.Overexpressed circR-ZC3HC1 or inhibited miR-384-5p expression promoted autophagy and inhibited apoptosis of OGD-treated neurons,which could be reversed by further 3-MA treatment.Mechanistically,circR-ZC3HC1 targeted miR-384-5p to mediate SIRT1 expression.miR-384-5p overexpression or SIRT1 knockdown in the presence of circR-ZC3HC1 overexpression in OGD-treated neurons lead to reduced autophagy and enhanced apoptosis.Conclusion:Collectively,circR-ZC3HC1 promoted neuronal autophagy to attenuate IS via miR-384-5p/SIRT1 axis.

Crosstalk among Oxidative Stress,Autophagy,and Apoptosis in the Protective Effects of Ginsenoside Rb1 on Brain Microvascular Endothelial Cells:A Mixed Computational and Experimental Study

Objective Brain microvascular endothelial cells (BMECs) were found to shift from their usually inactive state to an active state in ischemic stroke (IS) and cause neuronal damage. Ginsenoside Rb1 (GRb1),a component derived from medicinal plants,is known for its pharmacological benefits in IS,but its protective effects on BMECs have yet to be explored. This study aimed to investigate the potential protective effects of GRb1 on BMECs. Methods An in vitro oxygen-glucose deprivation/reperfusion (OGD/R) model was established to mimic ischemia-reperfusion (I/R) injury. Bulk RNA-sequencing data were analyzed by using the Human Autophagy Database and various bioinformatic tools,including gene set enrichment analysis (GSEA),Gene Ontology (GO) classification and enrichment analysis,Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis,protein-protein interaction network analysis,and molecular docking. Experimental validation was also performed to ensure the reliability of our findings. Results Rb1 had a protective effect on BMECs subjected to OGD/R injury. Specifically,GRb1 was found to modulate the interplay between oxidative stress,apoptosis,and autophagy in BMECs. Key targets such as sequestosome 1 (SQSTM1/p62),autophagy related 5 (ATG5),and hypoxia-inducible factor 1-alpha (HIF-1α) were identified,highlighting their potential roles in mediating the protective effects of GRb1 against IS-induced damage. Conclusion GRbl protects BMECs against OGD/R injury by influencing oxidative stress,apoptosis,and autophagy. The identification of SQSTM1/p62,ATG5,and HIF-1α as promising targets further supports the potential of GRb1 as a therapeutic agent for IS,providing a foundation for future research into its mechanisms and applications in IS treatment.

Salsolinol as an RNA m~6A methylation inducer mediates dopaminergic neuronal death by regulating YAP1 and autophagy

Salsolinol(1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline,Sal)is a catechol isoquinoline that causes neurotoxicity and shares structural similarity with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,an environmental toxin that causes Parkinson's disease.However,the mechanism by which Sal mediates dopaminergic neuronal death remains unclear.In this study,we found that Sal significantly enhanced the global level of N~6-methyladenosine(m~6A)RNA methylation in PC12 cells,mainly by inducing the downregulation of the expression of m~6A demethylases fat mass and obesity-associated protein(FTO)and alk B homolog 5(ALKBH5).RNA sequencing analysis showed that Sal downregulated the Hippo signaling pathway.The m~6A reader YTH domain-containing family protein 2(YTHDF2)promoted the degradation of m~6A-containing Yes-associated protein 1(YAP1)mRNA,which is a downstream key effector in the Hippo signaling pathway.Additionally,downregulation of YAP1 promoted autophagy,indicating that the mutual regulation between YAP1 and autophagy can lead to neurotoxicity.These findings reveal the role of Sal on m~6A RNA methylation and suggest that Sal may act as an RNA methylation inducer mediating dopaminergic neuronal death through YAP1 and autophagy.Our results provide greater insights into the neurotoxic effects of catechol isoquinolines compared with other studies and may be a reference for assessing the involvement of RNA methylation in the pathogenesis of Parkinson's disease.

DAPK2 promotes autophagy to accelerate the progression of ossification of the posterior longitudinal ligament through the mTORC1 complex

Background:Ossification of the posterior longitudinal ligament(OPLL)is a prevalent condition in orthopedics.While death-associated protein kinase 2(DAPK2)is known to play roles in cellular apoptosis and autophagy,its specific contributions to the advancement of OPLL are not well understood.Methods:Ligament fibroblasts were harvested from patients diagnosed with OPLL.Techniques such as real-time reverse transcriptasepolymerase chain reaction(RT-qPCR)and Western blot analysis were employed to assess DAPK2 levels in both ligament tissues and cultured fibroblasts.The extent of osteogenic differentiation in these cells was evaluated using an alizarin red S(ARS)staining.Additionally,the expression of ossification markers and autophagy markers was quantified.The autophagic activity was further analyzed through LC3 immunofluorescence and transmission electron microscopy(TEM).An in vivo heterotopic bone formation assay was conducted in mice to assess the role of DAPK2 in ossification.Results:Elevated DAPK2 expression was confirmed in both OPLL patient tissues and derived fibroblasts,in contrast to non-OPLL controls.Silencing of DAPK2 significantly curtailed osteogenic differentiation and autophagy in these fibroblasts,evidenced by decreased levels of LC3,and Beclin1,and reduced autophagosome formation.Additionally,DAPK2 was found to inhibit the mechanistic target of the rapamycin complex 1(mTORC1)complex’s activity.In vivo studies demonstrated that DAPK2 facilitates ossification,and this effect could be counteracted by the mTORC1 inhibitor rapamycin.Conclusion:DAPK2 enhances autophagy and osteogenic processes in OPLL through modulation of the mTORC1 pathway.

Corilagin alleviates podocyte injury in diabetic nephropathy by regulating autophagy via the SIRT1-AMPK pathway

BACKGROUND Diabetic nephropathy(DN)is the most frequent chronic microvascular consequence of diabetes,and podocyte injury and malfunction are closely related to the development of DN.Studies have shown that corilagin(Cor)has hepatoprotective,anti-inflammatory,antibacterial,antioxidant,anti-hypertensive,antidiabetic,and anti-tumor activities.AIM To explore the protective effect of Cor against podocyte injury in DN mice and the underlying mechanisms.METHODS Streptozotocin and a high-fat diet were combined to generate DN mice models,which were then divided into either a Cor group or a DN group(n=8 in each group).Mice in the Cor group were intraperitoneally injected with Cor(30 mg/kg/d)for 12 wk,and mice in the DN group were treated with saline.Biochemical analysis was used to measure the blood lipid profiles.Hematoxylin and eosin staining was used to detect pathological changes in kidney tissue.Immunohistochemistry and Western blotting were used to assess the protein expression of nephrin and podocin.Mouse podocyte cells(MPC5)were cultured and treated with glucose(5 mmol/L),Cor(50μM),high glucose(HG)(30 mmol/L),and HG(30 mmol/L)plus Cor(50μM).Real-time quantitative PCR and Western blotting RESULTS Compared with the control group,the DN mice models had increased fasting blood glucose,glycosylated hemoglobin,triglycerides,and total cholesterol,decreased nephrin and podocin expression,increased apoptosis rate,elevated inflammatory cytokines,and enhanced oxidative stress.All of the conditions mentioned above were alleviated after intervention with Cor.In addition,Cor therapy improved SIRT1 and AMPK expression(P<0.001),inhibited reactive oxygen species and oxidative stress,and elevated autophagy in HG-induced podocytes(P<0.01).CONCLUSION Cor alleviates podocyte injury by regulating autophagy via the SIRT1-AMPK pathway,thereby exerting its protective impact on renal function in DN mice.

Overexpression of TRPV1 activates autophagy in human lens epithelial cells under hyperosmotic stress through Ca^(2+)-dependent AMPK/mTOR pathway

●AIM:To explore whether autophagy functions as a cellular adaptation mechanism in lens epithelial cells(LECs)under hyperosmotic stress.●METHODS:LECs were treated with hyperosmotic stress at the concentration of 270,300,400,500,or 600 mOsm for 6,12,18,24h in vitro.Polymerase chain reaction(PCR)was employed for the mRNA expression of autophagyrelated genes,while Western blotting detected the targeted protein expression.The transfection of stub-RFP-sens-GFPLC3 autophagy-related double fluorescence lentivirus was conducted to detect the level of autophagy flux.Scanning electron microscopy was used to detect the existence of autolysosome.Short interfering RNA of autophagy-related gene(ATG)7,transient receptor potential vanilloid(TRPV)1 overexpression plasmid,related agonists and inhibitors were employed to their influence on autophagy related pathway.Flow cytometry was employed to test the apoptosis and intracellular Ca^(2+)level.Mitochondrial membrane potential was measured by JC-1 staining.The cell counting kit-8 assay was used to calculate the cellular viability.The wound healing assay was used to evaluate the wound closure rate.GraphPad 6.0 software was utilized to evaluate the data.●RESULTS:The hyperosmotic stress activated autophagy in a pressure-and time-dependent manner in LECs.Beclin 1 protein expression and conversion of LC3B II to LC3B I increased,whereas sequestosome-1(SQSTM1)protein expression decreased.Transient Ca^(2+)influx was stimulated caused by hyperosmotic stress,levels of mammalian target of rapamycin(mTOR)phosphorylation decreased,and the level of AMP-activated protein kinase(AMPK)phosphorylation increased in the early stage.Based on this evidence,autophagy activation through the Ca^(2+)-dependent AMPK/mTOR pathway might represent an adaptation process in LECs under hyperosmotic stress.Hyperosmotic stress decreased cellular viability and accelerated apoptosis in LECs and cellular migration decreased.Inhibition of autophagy by ATG7 knockdown had similar results.TRPV1 overexpression increased autophagy and might be crucial in the occurrence of autophagy promoted by hyperosmotic stress.●CONCLUSION:A combination of hyperosmotic stress and autophagy inhibition may be a promising approach to decrease the number of LECs in the capsular bag and pave the way for improving prevention of posterior capsular opacification and capsular fibrosis.

Low Selenium and Low Protein Exacerbate Myocardial Damage in Keshan Disease by Affecting the PINK1/Parkin-mediated Mitochondrial Autophagy Pathway

Objective Keshan disease(KD)is a myocardial mitochondrial disease closely related to insufficient selenium(Se)and protein intake.PTEN induced putative kinase 1(PINK1)/Parkin mediated mitochondrial autophagy regulates various physiological and pathological processes in the body.This study aimed to elucidate the relationship between PINK1/Parkin-regulated mitochondrial autophagy and KD-related myocardial injury.Methods A low Se and low protein animal model was established.One hundred Wistar rats were randomly divided into 5 groups(control group,low Se group,low protein group,low Se+low protein group,and corn from KD area group).The JC-1 method was used to detect the mitochondrial membrane potential(MMP).ELISA was used to detect serum creatine kinase MB(CK-MB),cardiac troponin I(cTnI),and mitochondrial-glutamicoxalacetic transaminase(M-GOT)levels.RT-PCR and Western blot analysis were used to detect the expression of PINK1,Parkin,sequestome 1(P62),and microtubule-associated proteins1A/1B light chain 3B(MAP1LC3B).Results The MMP was significantly decreased and the activity of CK-MB,cTnI,and M-GOT significantly increased in each experimental group(low Se group,low protein group,low Se+low protein group and corn from KD area group)compared with the control group(P<0.05 for all).The mRNA and protein expression levels of PINK1,Parkin and MAP1LC3B were profoundly increased,and those of P62 markedly decreased in the experimental groups compared with the control group(P<0.05 for all).Conclusion Low Se and low protein levels exacerbate myocardial damage in KD by affecting the PINK1/Parkin-mediated mitochondrial autophagy pathway.

MicroRNA-451 from Human Umbilical Cord-Derived Mesenchymal Stem Cell Exosomes Inhibits Alveolar Macrophage Autophagy via Tuberous Sclerosis Complex 1/Mammalian Target of Rapamycin Pathway to Attenuate Burn-Induced Acute Lung Injury in Rats

Objective Our previous studies established that microRNA(miR)-451 from human umbilical cord mesenchymal stem cell-derived exosomes(hUC-MSC-Exos)alleviates acute lung injury(ALI).This study aims to elucidate the mechanisms by which miR-451 in hUC-MSC-Exos reduces ALI by modulating macrophage autophagy.Methods Exosomes were isolated from hUC-MSCs.Severe burn-induced ALI rat models were treated with hUC-MSC-Exos carrying the miR-451 inhibitor.Hematoxylin-eosin staining evaluated inflammatory injury.Enzyme-linked immunosorbnent assay measured lipopolysaccharide(LPS),tumor necrosis factor-α,and interleukin-1βlevels.qRT-PCR detected miR-451 and tuberous sclerosis complex 1(TSC1)expressions.The regulatory role of miR-451 on TSC1 was determined using a dual-luciferase reporter system.Western blotting determined TSC1 and proteins related to the mammalian target of rapamycin(mTOR)pathway and autophagy.Immunofluorescence analysis was conducted to examine exosomes phagocytosis in alveolar macrophages and autophagy level.Results hUC-MSC-Exos with miR-451 inhibitor reduced burn-induced ALI and promoted macrophage autophagy.MiR-451 could be transferred from hUC-MSCs to alveolar macrophages via exosomes and directly targeted TSC1.Inhibiting miR-451 in hUC-MSC-Exos elevated TSC1 expression and inactivated the mTOR pathway in alveolar macrophages.Silencing TSC1 activated mTOR signaling and inhibited autophagy,while TSC1 knockdown reversed the autophagy from the miR-451 inhibitor-induced.Conclusion miR-451 from hUC-MSC exosomes improves ALI by suppressing alveolar macrophage autophagy through modulation of the TSC1/mTOR pathway,providing a potential therapeutic strategy for ALI.

Resolvin D1 Induces mTOR-independent and ATG5-dependent Autophagy in BV-2 Microglial Cells

Objective The activation state of microglia is known to occupy a central position in the pathophysiological process of cerebral inflammation.Autophagy is a catabolic process responsible for maintaining cellular homeostasis.In recent years,autophagy has been demonstrated to play an important role in neuroinflammation.Resolvin D1(RvD1)is a promising therapeutic mediator that has been shown to exert substantial anti-inflammatory and proresolving activities.However,whether RvD1-mediated resolution of inflammation in microglia is related to autophagy regulation needs further investigation.The present study aimed to explore the effect of RvD1 on microglial autophagy and its corresponding pathways.Methods Mouse microglial cells(BV-2)were cultured,treated with RvD1,and examined by Western blotting,confocal immunofluorescence microscopy,transmission electron microscopy,and flow cytometry.Results RvD1 promoted autophagy in both BV-2 cells and mouse primary microglia by favoring the maturation of autophagosomes and their fusion with lysosomes.Importantly,RvD1 had no significant effect on the activation of mammalian target of rapamycin(mTOR)signaling.Furthermore,RvD1-induced mTOR-independent autophagy was confirmed by observing reduced cytoplasmic calcium levels and suppressed calcium/calmodulin-dependent protein kinase II(CaMK II)activation.Moreover,by downregulating ATG5,the increased phagocytic activity induced by RvD1 was demonstrated to be tightly controlled by ATG5-dependent autophagy.Conclusion The present work identified a previously unreported mechanism responsible for the role of RvD1 in microglial autophagy,highlighting its therapeutic potential against neuroinflammation.

LIN28A attenuates high glucose-induced retinal pigmented epithelium injury through activating SIRT1-dependent autophagy

AIM:To evaluate the effects of LIN28A(human)on high glucose-induced retinal pigmented epithelium(RPE)cell injury and its possible mechanism.METHODS:Diabetic retinopathy model was generated following 48h of exposure to 30 mmol/L high glucose(HG)in ARPE-19 cells.Quantitative real-time polymerase chain reaction(qRT-PCR)and Western blot tested the expression of the corresponding genes and proteins.Cell viability as well as apoptosis was determined through cell counting kit-8(CCK-8)and flow cytometry assays.Immunofluorescence assay was adopted to evaluate autophagy activity.Caspase 3 activity,oxidative stress markers,and cytokines were appraised adopting their commercial kits,respectively.Finally,ARPE-19 cells were preincubated with EX527,a Sirtuin 1(SIRT1)inhibitor,prior to HG stimulation to validate the regulatory mechanism.RESULTS:LIN28A was downregulated in HG-challenged ARPE-19 cells.LIN28A overexpression greatly inhibited HGinduced ARPE-19 cell viability loss,apoptosis,oxidative damage as well as inflammatory response.Meanwhile,the repressed autophagy and SIRT1 in ARPE-19 cells challenged with HG were elevated after LIN28A overexpression.In addition,treatment of EX527 greatly inhibited the activated autophagy following LIN28A overexpression and partly abolished the protective role of LIN28A against HG-elicited apoptosis,oxidative damage as well as inflammation in ARPE-19 cells.CONCLUSION:LIN28A exerts a protective role against HG-elicited RPE oxidative damage,inflammation,as well as apoptosis via regulating SIRT1/autophagy.

MiR-873 regulates cell autophagy by targeting Beclin1 to promot inflammation and apoptosis of bronchial epithelial cells

Objective:Investigating the effects of miR-873 on apoptosis and autophagy in bronchial epithelial cells,as well as its regulatory role on Beclin1.Methods:Following transfection of miR-873 mimic into 16HBE cells for 48 hours,the mRNA level of miR-873 was quantified by qRT-PCR,and cell viability was evaluated by CCK-8 assay.The levels of IL-2,IL-6,IL-10,and TNF-αin the cell supernatant were determined using ELISA assay,while cell apoptosis was detected by TUNEL staining.LC-3 protein expression was examined by immunofluorescence,and mRNA and protein expression levels of Beclin1 were analyzed by qRT-PCR and Western blot,respectively.Moreover,dual-luciferase reporter gene technology was employed to investigate the binding site between miR-873 and Beclin1.Results:Transfection of miR-873 mimic into 16HBE cells significantly upregulated the mRNA level of miR-873,which led to the inhibition of cell proliferation and the promotion of secretion of pro-inflammatory cytokines IL-2,IL-6,and TNF-α,while suppressing the secretion of anti-inflammatory cytokine IL-10.Moreover,miR-873 induced cell apoptosis and inhibited the expression of LC-3.Dual-luciferase reporter gene assay further confirmed the presence of binding sites between miR-873 and Beclin1 gene.Besides,miR-873 could target and suppress the mRNA and protein expression levels of Beclin1.Conclusion:miR-873 might modulate cell autophagy by targeting the Beclin1 gene,which can potentially promote inflammation and apoptosis in bronchial epithelial cells.

High expression circRALGPS2 in atretic follicle induces chicken granulosa cell apoptosis and autophagy via encoding a new protein

Background The reproductive performance of chickens mainly depends on the development of follicles.Abnor-mal follicle development can lead to decreased reproductive performance and even ovarian disease among chick-ens.Chicken is the only non-human animal with a high incidence of spontaneous ovarian cancer.In recent years,the involvement of circRNAs in follicle development and atresia regulation has been confirmed.Results In the present study,we used healthy and atretic chicken follicles for circRNA RNC-seq.The results showed differential expression of circRALGPS2.It was then confirmed that circRALGPS2 can translate into a protein,named cir-cRALGPS2-212aa,which has IRES activity.Next,we found that circRALGPS2-212aa promotes apoptosis and autophagy in chicken granulosa cells by forming a complex with PARP1 and HMGB1.Conclusions Our results revealed that circRALGPS2 can regulate chicken granulosa cell apoptosis and autophagy through the circRALGPS2-212aa/PARP1/HMGB1 axis.

Fanlian Huazhuo Formula alleviates high-fat diet-induced nonalcoholic fatty liver disease by modulating autophagy and lipid synthesis signaling pathway

BACKGROUND Fanlian Huazhuo Formula(FLHZF)has the functions of invigorating spleen and resolving phlegm,clearing heat and purging turbidity.It has been identified to have therapeutic effects on type 2 diabetes mellitus(T2DM)in clinical application.Non-alcoholic fatty liver disease(NAFLD)is frequently diagnosed in patients with T2DM.However,the therapeutic potential of FLHZF on NAFLD and the underlying mechanisms need further investigation.AIM To elucidate the effects of FLHZF on NAFLD and explore the underlying hepatoprotective mechanisms in vivo and in vitro.METHODS HepG2 cells were treated with free fatty acid for 24 hours to induce lipid accumulation cell model.Subsequently,experiments were conducted with the different concentrations of freeze-dried powder of FLHZF for 24 hours.C57BL/6 mice were fed a high-fat diet for 8-week to establish a mouse model of NAFLD,and then treated with the different concentrations of FLHZF for 10 weeks.RESULTS FLHZF had therapeutic potential against lipid accumulation and abnormal changes in biochemical indicators in vivo and in vitro.Further experiments verified that FLHZF alleviated abnormal lipid metabolism might by reducing oxidative stress,regulating the AMPKα/SREBP-1C signaling pathway,activating autophagy,and inhibiting hepatocyte apoptosis.CONCLUSION FLHZF alleviates abnormal lipid metabolism in NAFLD models by regulating reactive oxygen species,autophagy,apoptosis,and lipid synthesis signaling pathways,indicating its potential for clinical application in NAFLD.

Novel insights into autophagy in gastrointestinal pathologies,mechanisms in metabolic dysfunction-associated fatty liver disease and acute liver failure

In this editorial,we comment on three articles published in a recent issue of World Journal of Gastroenterology.There is a pressing need for new research on autophagy's role in gastrointestinal(GI)disorders,and also novel insights into some liver conditions,such as metabolic dysfunction-associated fatty liver disease(MAFLD)and acute liver failure(ALF).Despite advancements,understanding autophagy's intricate mechanisms and implications in these diseases remains incomplete.Moreover,MAFLD's pathogenesis,encompassing hepatic steatosis and metabolic dysregulation,require further elucidation.Similarly,the mechanisms underlying ALF,a severe hepatic dysfunction,are poorly understood.Innovative studies exploring the interplay between autophagy and GI disorders,as well as defined mechanisms of MAFLD and ALF,are crucial for identifying therapeutic targets and enhancing diagnostic and treatment strategies to mitigate the global burden of these diseases.

Neuronal autophagy aggravates microglial inflammatory injury by downregulating CX3CL1/fractalkine after ischemic stroke

Ischemic stroke often induces excessive neuronal autophagy, resulting in brain damage; meanwhile, inflammatory responses stimulated by ischemia exacerbate neural injury. However, interactions between neuronal autophagy and microglial inflammation following ischemic stroke are poorly understood. CX3CL1/fractalkine, a membrane-bound chemokine expressed on neurons, can suppress microglial inflammation by binding to its receptor CX3CR1 on microglia. In the present study, to investigate whether autophagy could alter CX3CL1 expression on neurons and consequently change microglial inflammatory activity, middle cerebral artery occlusion(MCAO) was established in Sprague-Dawley rats to model ischemic stroke, and tissues from the ischemic penumbra were obtained to evaluate autophagy level and microglial inflammatory activity. MCAO rats were administered 3-methyladenine(autophagy inhibitor) or Tat-Beclin 1(autophagy inducer). Western blot assays were conducted to quantify expression of Beclin-1, nuclear factor kappa Bp65(NF-κB), light chain 3B(LC3B), and CX3CL1 in ischemic penumbra. Moreover, immunofluorescence staining was performed to quantify numbers of LC3B-, CX3CL1-, and Iba-1-positive cells in ischemic penumbra. In addition, enzyme linked immunosorbent assays were utilized to analyze concentrations of tumor necrosis factor alpha(TNF-α), interleukin 6(IL-6), interleukin 1 beta(IL-1β), and prostaglandin E2(PGE2). A dry/wet weight method was used to detect brain water content, while 2,3,5,-triphenyltetrazolium chloride staining was utilized to measure infarct volume. The results demonstrated that autophagy signaling(Beclin-1 and LC3B expression) in penumbra was prominently activated by MCAO, while CX3CL1 expression on autophagic neurons was significantly reduced and microglial inflammation was markedly activated. However, after inhibition of autophagy signaling with 3-methyladenine, CX3CL1 expression on neurons was obviously increased, whereas Iba-1 and NF-κB expression was downregulated; TNF-α, IL-6, IL-1β, and PGE2 levels were decreased; and cerebral edema was obviously mitigated. In contrast, after treatment with the autophagy inducer Tat-Beclin 1, CX3CL1 expression on neurons was further reduced; Iba-1 and NF-κB expression was increased; TNF-α, IL-6, IL-1β, and PGE2 levels were enhanced; and cerebral edema was aggravated. Our study suggests that ischemia-induced neuronal autophagy facilitates microglial inflammatory injury after ischemic stroke, and the efficacy of this process may be associated with downregulated CX3CL1 expression on autophagic neurons.

Hypoxia-induced Autophagy Contributes to Radioresistance via c-Jun-mediated Beclin1 Expression in Lung Cancer Cells

Reduced radiosensitivity of lung cancer cells represents a pivotal obstacle in clinical oncol- ogy. The hypoxia-inducible factor （HIF）-lα plays a crucial role in radiosensitivity, but the detailed mechanisms remain elusive. A relationship has been suggested to exist between hypoxia and autophagy recently. In the current study, we studied the effect of hypoxia-induced autophagy on radioresistance in lung cancer cell lines. A549 and H1299 cells were cultured under normoxia or hypoxia, followed by ir- radiation at dosage ranging from 0 to 8 Gy. Clonogenic assay was performed to calculate surviving frac- tion. EGFP-LC3 plasmid was stably transfected into cells to monitor autopbagic processes. Western blotting was used to evaluate the protein expression levels of HIF-lα, c-Jun, phosphorylated c-Jun, Be- clin 1, LC3 and p62. The mRNA levels of Beclin 1 were detected by qRT-PCR. We found that under hypoxia, both A549 and H1299 cells were radio-resistant compared with normoxia. Hypoxia-induced elevated HIF-1α protein expression preferentially triggered autophagy, accompanied by LC3 induction, EGFP-LC3 puncta and p62 degradation. In the meantime, HIF-1α increased downstream c-Jun phos- phorylation, which in turn upregulated Beclin 1 mRNA and protein expression. The upregulation of Be- clin 1 expression, instead of HIF-1α, could be blocked by SP600125 （a specific inhibitor of c-Jun NH2- terminal kinase）, followed by suppression of autophagy. Under hypoxia, combined treatment of irradia- tion and chloroquine （a potent autophagy inhibitor） significantly decreased the survival potential of lung cancer cells in vitro and in vivo. In conclusion, hypoxia-induced autophagy through evaluating Beclinl expression may be considered as a target to reverse the radioresistance in cancer cells.

Role of Beclin 1-dependent Autophagy in Cardioprotection of Ischemic Preconditioning

Emerging evidence indicates that ischemic preconditioning （IPC） induces autophagy which attenuates myocardial ischemia/reperfusion （I/R） injury. However, the precise mechanisms remain com- plex and unclear. The present study was to investigate which autophagy pathway was involved in the cardioprotection induced by IPC, so that we can acquire an attractive treatment way for iscbemic heart disease. Adult male Sprague-Dawley （SD） rats were randomly divided into sham group, I/R group and IPC group. IPC was induced with three cycles of 5 min regional ischemia alternating with 5 m^n reper- fusion in a heart I/R model. Samples were taken from the center of the infracted heart and examined by using the electron microscopy, the terminal deoxynucleotidyl transferase-mediated nick end-labeling （TUNEL） method, Western blotting and co-immunoprecipitation （Co-IP）. A large number of autophagic vacuoles were observed in the cardiomyocytes oflPC group as compared with I/R group. LC3-II forma- tion, an autophagy marker, was up-regulated in IPC group as compared with FR group （P〈0.05）. Moreover, the interaction between Beclin 1 and Bcl-2 was significantly increased in IPC group as com- pared with I/R group （P〈0.01）. It was also found that IPC decreased I/R-induced apoptosis （P〈0.01）. These results suggest that IPC inhibits Beclin 1-dependent excessive autophagy in reperfusion phase and cooperates with anti-apoptosis pathway to diminish the cell death induced by the myocardial I/R injury.