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1.
J Transl Med ; 22(1): 617, 2024 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-38961399

RESUMO

INTRODUCTION: Intrauterine adhesions (IUA) manifest as endometrial fibrosis, often causing infertility or recurrent miscarriage; however, their pathogenesis remains unclear. OBJECTIVES: This study assessed the role of Dickkopf WNT signaling pathway inhibitor 1 (DKK1) and autophagy in endometrial fibrosis, using clinical samples as well as in vitro and in vivo experiments. METHODS: Immunohistochemistry, immunofluorescence and western blot were used to determine the localization and expression of DKK1 in endometrium; DKK1 silencing and DKK1 overexpression were used to detect the biological effects of DKK1 silencing or expression in endometrial cells; DKK1 gene knockout mice were used to observe the phenotypes caused by DKK1 gene knockout. RESULTS: In patients with IUA, DKK1 and autophagy markers were down-regulated; also, α-SMA and macrophage localization were increased in the endometrium. DKK1 conditional knockout (CKO) mice showed a fibrotic phenotype with decreased autophagy and increased localization of α-SMA and macrophages in the endometrium. In vitro studies showed that DKK1 knockout (KO) suppressed the autophagic flux of endometrial stromal cells. In contrast, ectopic expression of DKK1 showed the opposite phenotype. Mechanistically, we discovered that DKK1 regulates autophagic flux through Wnt/ß-catenin and PI3K/AKT/mTOR pathways. Further studies showed that DKK1 KO promoted the secretion of interleukin (IL)-8 in exosomes, thereby promoting macrophage proliferation and metastasis. Also, in DKK1 CKO mice, treatment with autophagy activator rapamycin partially restored the endometrial fibrosis phenotype. CONCLUSION: Our findings indicated that DKK1 was a potential diagnostic marker or therapeutic target for IUA.


Assuntos
Autofagia , Endométrio , Exossomos , Fibrose , Peptídeos e Proteínas de Sinalização Intercelular , Macrófagos , Camundongos Knockout , Miofibroblastos , Animais , Feminino , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Endométrio/metabolismo , Endométrio/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Humanos , Exossomos/metabolismo , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Adulto
2.
Proc Natl Acad Sci U S A ; 121(28): e2404062121, 2024 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-38968109

RESUMO

Nutrient sensing and adaptation in the placenta are essential for pregnancy viability and proper fetal growth. Our recent study demonstrated that the placenta adapts to nutrient insufficiency through mechanistic target of rapamycin (mTOR) inhibition-mediated trophoblast differentiation toward syncytiotrophoblasts (STBs), a highly specialized multinucleated trophoblast subtype mediating extensive maternal-fetal interactions. However, the underlying mechanism remains elusive. Here, we unravel the indispensable role of the mTORC1 downstream transcriptional factor TFEB in STB formation both in vitro and in vivo. TFEB deficiency significantly impaired STB differentiation in human trophoblasts and placenta organoids. Consistently, systemic or trophoblast-specific deletion of Tfeb compromised STB formation and placental vascular construction, leading to severe embryonic lethality. Mechanistically, TFEB conferred direct transcriptional activation of the fusogen ERVFRD-1 in human trophoblasts and thereby promoted STB formation, independent of its canonical function as a master regulator of the autophagy-lysosomal pathway. Moreover, we demonstrated that TFEB directed the trophoblast syncytialization response driven by mTOR complex 1 (mTORC1) signaling. TFEB expression positively correlated with the reinforced trophoblast syncytialization in human fetal growth-restricted placentas exhibiting suppressed mTORC1 activity. Our findings substantiate that the TFEB-fusogen axis ensures proper STB formation during placenta development and under nutrient stress, shedding light on TFEB as a mechanistic link between nutrient-sensing machinery and trophoblast differentiation.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Diferenciação Celular , Alvo Mecanístico do Complexo 1 de Rapamicina , Trofoblastos , Trofoblastos/metabolismo , Humanos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Feminino , Gravidez , Camundongos , Animais , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Placenta/metabolismo , Transdução de Sinais , Autofagia/fisiologia
3.
PeerJ ; 12: e17619, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38952980

RESUMO

Background: Andrographolide (Andro), an extract of Andrographis paniculate (Burm.f.) Wall. ex Nees (Acanthaceae), possesses diverse biologically active properties. However, the precise mechanisms and effects of Andro on pancreatic cancer (PC) remain unclear. Methods: The cytotoxic potential of Andro and underlying mechanism towards PC cells was investigated through in vitro experiments and a xenograft mouse model. PC cells were first subjected to varying concentrations of Andro. The reactive oxygen species (ROS) was assessed using flow cytometry and DCFH-DA staining. The apoptosis rate was detected by flow cytometry. Additionally, western blot was applied to evaluate the expression levels of cleaved-caspase-3, DJ-1, LC3-I, LC3-II, and p62. To further elucidate the involvement of ROS accumulation and autophagy, we employed N-acetylcysteine as a scavenger of ROS and 3-Methyladenine as an inhibitor of autophagy. Results: Andro demonstrated potent anti-proliferative effects on PC cells and induced apoptosis, both in vitro and in vivo. The cytotoxicity of Andro on PC cells was counteracted by DJ-1 overexpression. The reduction in DJ-1 expression caused by Andro led to ROS accumulation, subsequently inhibiting the growth of PC cells. Furthermore, Andro stimulated cytoprotective autophagy, thus weakening the antitumor effect. Pharmacological blockade of autophagy further enhanced the antitumor efficacy of Andro. Conclusion: Our study indicated that ROS accumulation induced by the DJ-1 reduction played a key role in Andro-mediated PC cell inhibition. Furthermore, the protective autophagy induced by the Andro in PC cells is a mechanism that needs to be addressed in future studies.


Assuntos
Apoptose , Autofagia , Diterpenos , Neoplasias Pancreáticas , Proteína Desglicase DJ-1 , Espécies Reativas de Oxigênio , Espécies Reativas de Oxigênio/metabolismo , Diterpenos/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/metabolismo , Autofagia/efeitos dos fármacos , Proteína Desglicase DJ-1/metabolismo , Proteína Desglicase DJ-1/genética , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Apoptose/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Camundongos Nus
4.
Methods Mol Biol ; 2814: 97-106, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38954200

RESUMO

Autophagy is an intracellular clearance and recycling pathway that delivers different types of cargos to lysosomes for degradation. In recent years, autophagy has attracted considerable medical interest, and many different techniques are being developed to study this process in experimental models such as Dictyostelium. Here we describe the use of different autophagic markers in confocal microscopy, in vivo and also in fixed cells. In particular, we describe the use of the GFP-Atg8-RFP-Atg8ΔG marker and the optimization of the GFP-PgkA cleavage assay to detect small differences in autophagy flux.


Assuntos
Autofagia , Dictyostelium , Microscopia Confocal , Dictyostelium/metabolismo , Dictyostelium/fisiologia , Autofagia/fisiologia , Microscopia Confocal/métodos , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/genética , Lisossomos/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética
5.
Cell Biol Toxicol ; 40(1): 51, 2024 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-38958792

RESUMO

The implementation of Zinc oxide nanoparticles (ZnO NPs) raises concerns regarding their potential toxic effects on human health. Although more and more researches have confirmed the toxic effects of ZnO NPs, limited attention has been given to their impact on the early embryonic nervous system. This study aimed to explore the impact of exposure to ZnO NPs on early neurogenesis and explore its underlying mechanisms. We conducted experiments here to confirm the hypothesis that exposure to ZnO NPs causes neural tube defects in early embryonic development. We first used mouse and chicken embryos to confirm that ZnO NPs and the Zn2+ they release are able to penetrate the placental barrier, influence fetal growth and result in incomplete neural tube closure. Using SH-SY5Y cells, we determined that ZnO NPs-induced incomplete neural tube closure was caused by activation of various cell death modes, including ferroptosis, apoptosis and autophagy. Moreover, dissolved Zn2+ played a role in triggering widespread cell death. ZnO NPs were accumulated within mitochondria after entering cells, damaging mitochondrial function and resulting in the over production of reactive oxygen species, ultimately inducing cellular oxidative stress. The N-acetylcysteine (NAC) exhibits significant efficacy in mitigating cellular oxidative stress, thereby alleviating the cytotoxicity and neurotoxicity brought about by ZnO NPs. These findings indicated that the exposure of ZnO NPs in early embryonic development can induce cell death through oxidative stress, resulting in a reduced number of cells involved in early neural tube closure and ultimately resulting in incomplete neural tube closure during embryo development. The findings of this study could raise public awareness regarding the potential risks associated with the exposure and use of ZnO NPs in early pregnancy.


Assuntos
Desenvolvimento Embrionário , Defeitos do Tubo Neural , Tubo Neural , Estresse Oxidativo , Espécies Reativas de Oxigênio , Óxido de Zinco , Óxido de Zinco/toxicidade , Animais , Estresse Oxidativo/efeitos dos fármacos , Embrião de Galinha , Desenvolvimento Embrionário/efeitos dos fármacos , Camundongos , Tubo Neural/efeitos dos fármacos , Tubo Neural/embriologia , Tubo Neural/metabolismo , Humanos , Defeitos do Tubo Neural/induzido quimicamente , Defeitos do Tubo Neural/metabolismo , Defeitos do Tubo Neural/embriologia , Defeitos do Tubo Neural/patologia , Espécies Reativas de Oxigênio/metabolismo , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Feminino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Nanopartículas Metálicas/toxicidade , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Nanopartículas/toxicidade
6.
Sci Rep ; 14(1): 15133, 2024 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-38956194

RESUMO

The goal of this study was to evaluate the intensity of autophagy and ubiquitin-dependent proteolysis processes occurring in myocardium of left ventricle (LV) in subsequent stages of pulmonary arterial hypertension (PAH) to determine mechanisms responsible for LV mass loss in a monocrotaline-induced PAH rat model. LV myocardium samples collected from 32 Wistar rats were analyzed in an early PAH group (n = 8), controls time-paired (n = 8), an end-stage PAH group (n = 8), and their controls (n = 8). Samples were subjected to histological analyses with immunofluorescence staining, autophagy assessment by western blotting, and evaluation of ubiquitin-dependent proteolysis in the LV by immunoprecipitation of ubiquitinated proteins. Echocardiographic, hemodynamic, and heart morphometric parameters were assessed regularly throughout the experiment. Considerable morphological and hemodynamic remodeling of the LV was observed over the course of PAH. The end-stage PAH was associated with significantly impaired LV systolic function and a decrease in LV mass. The LC3B-II expression in the LV was significantly higher in the end-stage PAH group compared to the early PAH group (p = 0.040). The measured LC3B-II/LC3B-I ratios in the end-stage PAH group were significantly elevated compared to the controls (p = 0.039). Immunofluorescence staining showed a significant increase in the abundance of LC3 puncta in the end-stage PAH group compared to the matched controls. There were no statistically significant differences in the levels of expression of all ubiquitinated proteins when comparing both PAH groups and matched controls. Autophagy may be considered as the mechanism behind the LV mass loss at the end stage of PAH.


Assuntos
Autofagia , Ventrículos do Coração , Proteólise , Hipertensão Arterial Pulmonar , Ratos Wistar , Ubiquitina , Animais , Ubiquitina/metabolismo , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Ratos , Masculino , Hipertensão Arterial Pulmonar/metabolismo , Hipertensão Arterial Pulmonar/patologia , Modelos Animais de Doenças , Miocárdio/metabolismo , Miocárdio/patologia , Ecocardiografia , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Remodelação Ventricular
7.
Cell Mol Biol Lett ; 29(1): 95, 2024 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-38956466

RESUMO

BACKGROUND: An increasing number of studies have demonstrated the association of circular RNAs (circRNAs) with the pathological processes of various diseases and their involvement in the onset and progression of multiple cancers. Nevertheless, the functional roles and underlying mechanisms of circRNAs in the autophagy regulation of gastric cancer (GC) have not been fully elucidated. METHODS: We used transmission electron microscopy and the mRFP-GFP-LC3 dual fluorescent autophagy indicator to investigate autophagy regulation. The cell counting kit-8 assay, colony formation assay, 5-ethynyl-2'-deoxyuridine incorporation assay, Transwell assay, and Western blot assay were conducted to confirm circPTPN22's influence on GC progression. Dual luciferase reporter assays validated the binding between circPTPN22 and miR-6788-5p, as well as miR-6788-5p and p21-activated kinase-1 (PAK1). Functional rescue experiments assessed whether circPTPN22 modulates PAK1 expression by competitively binding miR-6788-5p, affecting autophagy and other biological processes in GC cells. We investigated the impact of circPTPN22 on in vivo GC tumors using a nude mouse xenograft model. Bioinformatics tools predicted upstream regulatory transcription factors and binding proteins of circPTPN22, while chromatin immunoprecipitation and ribonucleoprotein immunoprecipitation assays confirmed the binding status. RESULTS: Upregulation of circPTPN22 in GC has been shown to inhibit autophagy and promote cell proliferation, migration, and invasion. Mechanistically, circPTPN22 directly binds to miR-6788-5p, subsequently regulating the expression of PAK1, which activates protein kinase B (Akt) and extracellular signal-regulated kinase (Erk) phosphorylation. This modulation ultimately affects autophagy levels in GC cells. Additionally, runt-related transcription factor 1 (RUNX1) negatively regulates circPTPN22 expression, while RNA-binding proteins such as FUS (fused in sarcoma) and ELAVL1 (recombinant ELAV-like protein 1) positively regulate its expression. Inhibition of the autophagy pathway can increase FUS expression, further upregulating circPTPN22 in GC cells, thereby exacerbating the progression of GC. CONCLUSION: Under the regulation of the transcription factor RUNX1 and RNA-binding proteins FUS and ELAVL1, circPTPN22 activates the phosphorylation of Akt and Erk through the miR-6788-5p/PAK1 axis, thereby modulating autophagy in GC cells. Inhibition of autophagy increases FUS, which in turn upregulates circPTPN22, forming a positive feedback loop that ultimately accelerates the progression of GC.


Assuntos
Autofagia , Movimento Celular , Proliferação de Células , Subunidade alfa 2 de Fator de Ligação ao Core , Proteína Semelhante a ELAV 1 , MicroRNAs , RNA Circular , Proteína FUS de Ligação a RNA , Neoplasias Gástricas , Quinases Ativadas por p21 , Humanos , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Neoplasias Gástricas/metabolismo , RNA Circular/genética , RNA Circular/metabolismo , Autofagia/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Quinases Ativadas por p21/metabolismo , Quinases Ativadas por p21/genética , Proliferação de Células/genética , Proteína FUS de Ligação a RNA/metabolismo , Proteína FUS de Ligação a RNA/genética , Movimento Celular/genética , Linhagem Celular Tumoral , Animais , Proteína Semelhante a ELAV 1/metabolismo , Proteína Semelhante a ELAV 1/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Regulação Neoplásica da Expressão Gênica , Camundongos Nus , Camundongos , Invasividade Neoplásica , Camundongos Endogâmicos BALB C
8.
Mol Brain ; 17(1): 42, 2024 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-38956588

RESUMO

Glioblastoma (GBM) is an aggressive nervous system tumor with a poor prognosis. Although, surgery, radiation therapy, and chemotherapy are the current standard protocol for GBM patients, there is still a poor prognosis in these patients. Temozolomide (TMZ) as a first-line therapeutic agent in GBM can easily cross from the blood-brain barrier to inhibit tumor cell proliferation. However, there is a high rate of TMZ resistance in GBM patients. Since, there are limited therapeutic choices for GBM patients who develop TMZ resistance; it is required to clarify the molecular mechanisms of chemo resistance to introduce the novel therapeutic targets. MicroRNAs (miRNAs) regulate chemo resistance through regulation of drug metabolism, absorption, DNA repair, apoptosis, and cell cycle. In the present review we discussed the role of miRNAs in TMZ response of GBM cells. It has been reported that miRNAs mainly induced TMZ sensitivity by regulation of signaling pathways and autophagy in GBM cells. Therefore, miRNAs can be used as the reliable diagnostic/prognostic markers in GBM patients. They can also be used as the therapeutic targets to improve the TMZ response in GBM cells.


Assuntos
Neoplasias Encefálicas , Resistencia a Medicamentos Antineoplásicos , Glioblastoma , MicroRNAs , Temozolomida , Humanos , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Glioblastoma/genética , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Animais , Dacarbazina/análogos & derivados , Dacarbazina/uso terapêutico , Dacarbazina/farmacologia , Autofagia/efeitos dos fármacos , Autofagia/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos
9.
BMC Biol ; 22(1): 146, 2024 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-38956599

RESUMO

BACKGROUND: Metabolic associated fatty liver disease (MAFLD), a prevalent liver disorder affecting one-third of the global population, encompasses a spectrum ranging from fatty liver to severe hepatic steatosis. Both genetic and lifestyle factors, particularly diet and nutrition, contribute to its etiology. Folate deficiency, a frequently encountered type of malnutrition, has been associated with the pathogenesis of MAFLD and shown to impact lipid deposition. However, the underlying mechanisms of this relationship remain incompletely understood. We investigated the impact of disturbed folate-mediated one-carbon metabolism (OCM) on hepatic lipid metabolism both in vitro using human hepatoma cells and in vivo using transgenic fluorescent zebrafish displaying extent-, stage-, and duration-controllable folate deficiency upon induction. RESULTS: Disturbed folate-mediated one-carbon metabolism, either by inducing folate deficiency or adding anti-folate drug, compromises autophagy and causes lipid accumulation in liver cells. Disturbed folate status down-regulates cathepsin L, a key enzyme involved in autophagy, through inhibiting mTOR signaling. Interfered mitochondrial biology, including mitochondria relocation and increased fusion-fission dynamics, also occurs in folate-deficient hepatocytes. Folate supplementation effectively mitigated the impaired autophagy and lipid accumulation caused by the inhibition of cathepsin L activity, even when the inhibition was not directly related to folate deficiency. CONCLUSIONS: Disruption of folate-mediated OCM diminishes cathepsin L expression and impedes autophagy via mTOR signaling, leading to lipid accumulation within hepatocytes. These findings underscore the crucial role of folate in modulating autophagic processes and regulating lipid metabolism in the liver.


Assuntos
Autofagia , Ácido Fólico , Hepatócitos , Homeostase , Metabolismo dos Lipídeos , Peixe-Zebra , Autofagia/fisiologia , Ácido Fólico/metabolismo , Humanos , Hepatócitos/metabolismo , Animais , Deficiência de Ácido Fólico/metabolismo
10.
Stem Cell Res Ther ; 15(1): 189, 2024 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-38956646

RESUMO

BACKGROUND: Recent studies have proved the role of autophagy in mesenchymal stem cell (MSCs) function and regenerative properties. How and by which mechanism autophagy modulation can affect the juxtacrine interaction of MSCs should be addressed. Here, the role of autophagy was investigated in the formation of tunneling nanotubes (TNTs) and homotypic mitochondrial donation. METHODS: MSCs were incubated with 15 µM Metformin (Met) and/or 3 µM 3-methyladenine (3-MA) for 48 h. The formation of TNTs was assessed using bright-field and SEM images. The mitochondria density and ΔΨ values were monitored using flow cytometry analysis. Using RT-PCR and protein array, the close interaction and shared mediators between autophagy, apoptosis, and Wnt signaling pathways were also monitored. The total fatty acid profile was assessed using gas chromatography. RESULT: Data indicated the increase of TNT length and number, along with other cell projections after the induction of autophagy while these features were blunted in 3-MA-treated MSCs (p < 0.05). Western blotting revealed the significant reduction of Rab8 and p-FAK in 3-MA-treated MSCs (p < 0.05), indicating the inhibition of TNT assembly and vesicle transport. Likewise, the stimulation of autophagy increased autophagic flux and mitochondrial membrane integrity compared to 3-MA-treated MSCs. Despite these findings, protein levels of mitochondrial membrane Miro1 and 2 were unchanged after autophagy inhibition/stimulation (p > 0.05). We found that the inhibition/stimulation of autophagy can affect the protein, and transcription levels of several mediators related to Wnt and apoptosis signaling pathways involved in different cell bioactivities. Data confirmed the profound increase of mono and polyunsaturated/saturated fatty acid ratio in MSCs exposed to autophagy stimulator. CONCLUSIONS: In summary, autophagy modulation could affect TNT formation which is required for homotypic mitochondrial donation. Thus, the modulation of autophagy creates a promising perspective to increase the efficiency of cell-based therapies.


Assuntos
Autofagia , Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Mitocôndrias/metabolismo , Adenina/farmacologia , Adenina/análogos & derivados , Humanos , Nanotubos/química , Apoptose/efeitos dos fármacos , Animais , Metformina/farmacologia , Células Cultivadas , Via de Sinalização Wnt/efeitos dos fármacos , Estruturas da Membrana Celular
11.
J Orthop Surg Res ; 19(1): 387, 2024 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-38956661

RESUMO

Spinal cord injury (SCI) is a severe condition with an extremely high disability rate. It is mainly manifested as the loss of motor, sensory and autonomic nerve functions below the injury site. High-frequency transcranial magnetic stimulation, a recently developed neuromodulation method, can increase motor function in mice with spinal cord injury. This study aimed to explore the possible mechanism by which transcranial magnetic stimulation (TMS) restores motor function after SCI. A complete T8 transection model of the spinal cord was established in mice, and the mice were treated daily with 15 Hz high-frequency transcranial magnetic stimulation. The BMS was used to evaluate the motor function of the mice after SCI. Western blotting and immunofluorescence were used to detect the expression of Connexin43 (CX43) and autophagy-related proteins in vivo and in vitro, and correlation analysis was performed to study the relationships among autophagy, CX43 and motor function recovery after SCI in mice. Western blotting was used to observe the effect of magnetic stimulation on the expression of mTOR pathway members. In the control group, the expression of CX43 was significantly decreased, and the expression of microtubule-associated protein 1 A/1b light chain 3 (LC3II) and P62 was significantly increased after 4 weeks of spinal cord transection. After high-frequency magnetic stimulation, the level of CX43 decreased, and the levels of LC3II and P62 increased in primary astrocytes. The BMS of the magnetic stimulation group was greater than that of the control group. High-frequency magnetic stimulation can inhibit the expression of CX43, which negatively regulates autophagic flux. HF-rTMS increased the expression levels of mTOR, p-mTOR and p-S6. Our experiments showed that rTMS can restore hindlimb motor function in mice after spinal cord injury via regulation of the Cx43-autophagy loop and activation of the mTOR signalling pathway.


Assuntos
Autofagia , Conexina 43 , Recuperação de Função Fisiológica , Traumatismos da Medula Espinal , Estimulação Magnética Transcraniana , Animais , Estimulação Magnética Transcraniana/métodos , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/terapia , Recuperação de Função Fisiológica/fisiologia , Conexina 43/metabolismo , Autofagia/fisiologia , Camundongos , Serina-Treonina Quinases TOR/metabolismo , Camundongos Endogâmicos C57BL , Atividade Motora/fisiologia , Modelos Animais de Doenças , Masculino , Feminino
12.
Mol Plant Pathol ; 25(7): e13489, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38956897

RESUMO

A cell death pathway, ferroptosis, occurs in conidial cells and is critical for formation and function of the infection structure, the appressorium, in the rice blast fungus Magnaporthe oryzae. In this study, we identified an orthologous lysophosphatidic acid acyltransferase (Lpaat) acting at upstream of phosphatidylethanolamines (PEs) biosynthesis and which is required for such fungal ferroptosis and pathogenicity. Two PE species, DOPE and SLPE, that depend on Lpaat function for production were sufficient for induction of lipid peroxidation and the consequent ferroptosis, thus positively regulating fungal pathogenicity. On the other hand, both DOPE and SLPE positively regulated autophagy. Loss of the LPAAT gene led to a decrease in the lipidated form of the autophagy protein Atg8, which is probably responsible for the autophagy defect of the lpaatΔ mutant. GFP-Lpaat was mostly localized on the membrane of lipid droplets (LDs) that were stained by the fluorescent dye monodansylpentane (MDH), suggesting that LDs serve as a source of lipids for membrane PE biosynthesis and probably as a membrane source of autophagosome. Overall, our results reveal novel intracellular membrane-bound organelle dynamics based on Lpaat-mediated lipid metabolism, providing a temporal and spatial link of ferroptosis and autophagy.


Assuntos
Autofagia , Ferroptose , Oryza , Fosfatidiletanolaminas , Doenças das Plantas , Fosfatidiletanolaminas/metabolismo , Oryza/microbiologia , Oryza/metabolismo , Doenças das Plantas/microbiologia , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Aciltransferases/metabolismo , Aciltransferases/genética , Ascomicetos/patogenicidade , Ascomicetos/metabolismo
13.
Front Immunol ; 15: 1321657, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38975346

RESUMO

Tuberculosis (TB) remains a significant global health challenge, with approximately 1.5 million deaths per year. The Bacillus Calmette-Guérin (BCG) vaccine against TB is used in infants but shows variable protection. Here, we introduce a novel approach using a double gene knockout mutant (DKO) from wild-type Mycobacterium tuberculosis (Mtb) targeting fbpA and sapM genes. DKO exhibited enhanced anti-TB gene expression in mouse antigen-presenting cells, activating autophagy and inflammasomes. This heightened immune response improved ex vivo antigen presentation to T cells. Subcutaneous vaccination with DKO led to increased protection against TB in wild-type C57Bl/6 mice, surpassing the protection observed in caspase 1/11-deficient C57Bl/6 mice and highlighting the critical role of inflammasomes in TB protection. The DKO vaccine also generated stronger and longer-lasting protection than the BCG vaccine in C57Bl/6 mice, expanding both CD62L-CCR7-CD44+/-CD127+ effector T cells and CD62L+CCR7+/-CD44+CD127+ central memory T cells. These immune responses correlated with a substantial ≥ 1.7-log10 reduction in Mtb lung burden. The DKO vaccine represents a promising new approach for TB immunization that mediates protection through autophagy and inflammasome pathways.


Assuntos
Macrófagos , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis , Vacinas contra a Tuberculose , Tuberculose , Animais , Mycobacterium tuberculosis/imunologia , Camundongos , Macrófagos/imunologia , Tuberculose/imunologia , Tuberculose/prevenção & controle , Vacinas contra a Tuberculose/imunologia , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/genética , Inflamassomos/imunologia , Feminino , Vacina BCG/imunologia , Autofagia/imunologia , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/genética , Modelos Animais de Doenças
14.
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue ; 36(6): 616-623, 2024 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-38991961

RESUMO

OBJECTIVE: To investigate whether 6-shogaol (6-SH) alleviates oxygen-glucose deprivation/reoxygenation (OGD/R)-induced neuronal autophagy and calcium overload by promoting the expression of microRNA-26a-5p (miR-26a-5p) and inhibiting death-associated protein kinase 1 (DAPK1), and to explore its potential mechanisms. METHODS: Primary cultured logarithmic growth phase mouse hippocampal neurons HT22 cells were taken and cell counting kit-8 (CCK-8) was used to detect cell viability, searching for the optimal concentration of Na2S2O4. HT22 cells were divided into blank control group (NC group), OGD/R group (sugar-free culture medium + 10 mmol/L Na2S2O4 treatment for 1.5 hours followed by normal culture medium for 4 hours), 6-SH intervention group (cultured with 10 µmol/L 6-SH for 4 hours after OGD), negative control inhibitor pretreatment group (transfected with negative control inhibitor for 48 hours followed by OGD, then cultured with 6-SH for 4 hours), and miR-26a-5p inhibitor pretreatment group (transfected with miR-26a-5p inhibitor for 48 hours followed by OGD, then cultured with 6-SH for 4 hours). Cell viability of each group was detected by CCK-8 method; cell ultrastructure was observed under transmission electron microscopy; real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the gene expressions of DAPK1 and miR-26a-5p; molecular docking were used to verify the interaction between 6-SH and miR-26a-5p; dual-luciferase assay was used to verify the targeting relationship between DAPK1 and miR-26a-5p; flow cytometry was used to determine the levels of intracellular Ca2+; Western blotting was used to detect the protein expressions of phosphorylated-glutamate receptor 2B (p-NMDAR2B) Ser1303, DAPK1, autophagy related protein Beclin1, light chain 3 (LC3), and p-DAPK1 Ser308; immunofluorescence was used to detect the expression of LC3 and Beclin1. RESULTS: The results of the CCK-8 assay showed that the cell viability of the 6-SH intervention group was significantly increased compared to the OGD/R group, while the cell viability of the miR-26a-5p inhibitor pretreatment group was significantly decreased compared to the 6-SH intervention group. Transmission electron microscopy revealed that the number of autophagosomes in the 6-SH intervention group was significantly reduced compared to the OGD/R group, while the number of autophagosomes in the miR-26a-5p inhibitor pretreatment group was significantly increased compared to the 6-SH intervention group. RT-qPCR results showed that compared with the OGD/R group, the expression of miR-26a-5p was significantly upregulated and the expression of DAPK1 mRNA was significantly downregulated in the 6-SH intervention group; compared with the 6-SH intervention group, the expression of miR-26a-5p was significantly downregulated and the expression of DAPK1 mRNA was significantly upregulated in the miR-26a-5p inhibitor pretreatment group. Molecular docking verified the interaction between 6-SH and miR-26a-5p. Dual-luciferase reporter gene assay showed that compared with the negative control group, mmu-miR-26a-5p significantly downregulated the luciferase expression of m-DAPK1-3UTR-WT, indicating a binding interaction between them. Flow cytometry results showed that compared with the OGD/R group, the level of intracellular Ca2+; was significantly decreased in the 6-SH intervention group; compared with the 6-SH intervention group, the level of Ca2+ was significantly increased in the miR-26a-5p inhibitor pretreatment group. Western blotting results showed that compared with the OGD/R group, the protein expressions of p-NMDAR2B Ser1303, DAPK1, Beclin1, and LC3 were significantly decreased in the 6-SH intervention group (p-NMDAR2B Ser1303/ß-actin: 2.34±0.27 vs. 4.78±0.39, DAPK1/ß-actin: 1.40±0.13 vs. 2.37±0.21, Beclin1/ß-actin: 2.61±0.32 vs. 4.32±0.29, LC3/ß-actin: 2.52±0.45 vs. 5.09±0.18, all P < 0.05), while the protein expression of p-DAPK1 Ser308 was significantly increased (p-DAPK1 Ser308/ß-actin: 0.66±0.09 vs. 0.40±0.02, P < 0.05); compared with the 6-SH intervention group, the protein expressions of p-NMDAR2B Ser1303, DAPK1, Beclin1, and LC3 were significantly increased in the miR-26a-5p inhibitor pretreatment group (p-NMDAR2B Ser1303/ß-actin: 4.08±0.14 vs. 2.34±0.27, DAPK1/ß-actin: 1.96±0.15 vs. 1.40±0.13, Beclin1/ß-actin: 3.92±0.31 vs. 2.61±0.32, LC3/ß-actin: 4.33±0.33 vs. 2.52±0.45, all P < 0.05), while the expression of p-DAPK1 Ser308 protein was significantly decreased (p-DAPK1 Ser308/ß-actin: 0.33±0.12 vs. 0.66±0.09, P < 0.05); immunofluorescence staining showed that compared with the OGD/R group, the fluorescence intensity of LC3 and Beclin1 was significantly decreased in the 6-SH intervention group; compared with the 6-SH intervention group, the fluorescence intensity of LC3 and Beclin1 was significantly increased in the miR-26a-5p inhibitor pretreatment group. CONCLUSIONS: 6-SH can alleviate neuronal damage by regulating miR-26a-5p/DAPK1 to reduce autophagy and calcium overload in cells.


Assuntos
Autofagia , Proteínas Quinases Associadas com Morte Celular , MicroRNAs , Traumatismo por Reperfusão , MicroRNAs/genética , Animais , Camundongos , Proteínas Quinases Associadas com Morte Celular/metabolismo , Proteínas Quinases Associadas com Morte Celular/genética , Autofagia/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Isquemia Encefálica/metabolismo , Catecóis/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Hipocampo/metabolismo , Glucose
15.
Int J Mol Med ; 54(3)2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38994772

RESUMO

It is considered that the etiology of endometriosis is retrograde menstruation of endometrial tissue. Although shed endometrial cells are constantly exposed to a challenging environment with iron overload, oxidative stress and hypoxia, a few cells are able to survive and continue to proliferate and invade. Ferroptosis, an iron­dependent form of non­apoptotic cell death, is known to play a major role in the development and course of endometriosis. However, few papers have concentrated on the dynamic interaction between autophagy and ferroptosis throughout the progression of diseases. The present review summarized the current understanding of the mechanisms underlying autophagy and ferroptosis in endometriosis and discuss their role in disease development and progression. For the present narrative review electronic databases including PubMed and Google Scholar were searched for literature published up to the October 31, 2023. Autophagy and ferroptosis may be activated at early stages in endometriosis development. On the other hand, excessive activation of intrinsic pathways (e.g., estrogen and mechanistic target of rapamycin) may promote disease progression through autophagy inhibition. Furthermore, suppression of ferroptosis may cause further progression of endometriotic lesions. In conclusion, the autophagy and ferroptosis pathways may play a dual role in disease initiation and progression. The present review discussed the temporal transition of non­apoptotic cell death regulation during disease progression from retrograde endometrium to early lesions to established lesions.


Assuntos
Autofagia , Endometriose , Ferroptose , Humanos , Endometriose/metabolismo , Endometriose/patologia , Autofagia/fisiologia , Feminino , Animais , Cistos/patologia , Cistos/metabolismo , Endométrio/metabolismo , Endométrio/patologia
16.
Mol Med Rep ; 30(3)2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38994776

RESUMO

Cordycepin is a nucleoside molecule found in Cordyceps sinensis and can be obtained through chemical synthesis and biotransformation. Cordycepin has been extensively studied and has been shown to have antitumour activity. This activity includes effects on the autophagy process and inhibition of the MAPK/ERK and Hedgehog pathways. Ultimately, the inhibitory effect of cordycepin on tumour cells is due to the interplay of these effects. Cordycepin was shown to enhance the therapeutic effects of radiotherapy. There is increasing evidence indicating that cordycepin plays an anticancer role in the treatment of various cancers. The present review aims to provide a clear understanding of the antitumour mechanisms of cordycepin and discuss its present application in the treatment of tumours. This information can be an important theoretical basis and provide clinical guidance for the further development of cordycepin as an antitumour drug.


Assuntos
Desoxiadenosinas , Neoplasias , Humanos , Desoxiadenosinas/uso terapêutico , Desoxiadenosinas/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Animais , Antineoplásicos/uso terapêutico , Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
17.
J Cell Biol ; 223(9)2024 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-38980288

RESUMO

Autophagy is essential for maintaining glucose homeostasis. However, the mechanism by which cells sense and respond to glucose starvation to induce autophagy remains incomplete. Here, we show that calcium serves as a fundamental triggering signal that connects environmental sensing to the formation of the autophagy initiation complex during glucose starvation. Mechanistically, glucose starvation instigates the release of vacuolar calcium into the cytoplasm, thus triggering the activation of Rck2 kinase. In turn, Rck2-mediated Atg11 phosphorylation enhances Atg11 interactions with Bmh1/2 bound to the Snf1-Sip1-Snf4 complex, leading to recruitment of vacuolar membrane-localized Snf1 to the PAS and subsequent Atg1 activation, thereby initiating autophagy. We also identified Glc7, a protein phosphatase-1, as a critical regulator of the association between Bmh1/2 and the Snf1 complex. We thus propose that calcium-triggered Atg11-Bmh1/2-Snf1 complex assembly initiates autophagy by controlling Snf1-mediated Atg1 activation in response to glucose starvation.


Assuntos
Autofagia , Cálcio , Glucose , Proteínas Serina-Treonina Quinases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Glucose/metabolismo , Cálcio/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Fosforilação , Vacúolos/metabolismo , Vacúolos/genética
18.
Front Endocrinol (Lausanne) ; 15: 1404697, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38982993

RESUMO

Adipose tissue, an indispensable organ, fulfils the pivotal role of energy storage and metabolism and is instrumental in maintaining the dynamic equilibrium of energy and health of the organism. Adipocyte hypertrophy and adipocyte hyperplasia (adipogenesis) are the two primary mechanisms of fat deposition. Mature adipocytes are obtained by differentiating mesenchymal stem cells into preadipocytes and redifferentiation. However, the mechanisms orchestrating adipogenesis remain unclear. Autophagy, an alternative cell death pathway that sustains intracellular energy homeostasis through the degradation of cellular components, is implicated in regulating adipogenesis. Furthermore, adipose tissue functions as an endocrine organ, producing various cytokines, and certain inflammatory factors, in turn, modulate autophagy and adipogenesis. Additionally, autophagy influences intracellular redox homeostasis by regulating reactive oxygen species, which play pivotal roles in adipogenesis. There is a growing interest in exploring the involvement of autophagy, inflammation, and oxidative stress in adipogenesis. The present manuscript reviews the impact of autophagy, oxidative stress, and inflammation on the regulation of adipogenesis and, for the first time, discusses their interactions during adipogenesis. An integrated analysis of the role of autophagy, inflammation and oxidative stress will contribute to elucidating the mechanisms of adipogenesis and expediting the exploration of molecular targets for treating obesity-related metabolic disorders.


Assuntos
Adipogenia , Autofagia , Inflamação , Estresse Oxidativo , Adipogenia/fisiologia , Humanos , Autofagia/fisiologia , Estresse Oxidativo/fisiologia , Inflamação/metabolismo , Inflamação/patologia , Animais , Adipócitos/metabolismo , Adipócitos/patologia , Obesidade/metabolismo , Obesidade/patologia , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia
19.
Int J Nanomedicine ; 19: 6777-6809, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38983131

RESUMO

Chloroquine is a common antimalarial drug and is listed in the World Health Organization Standard List of Essential Medicines because of its safety, low cost and ease of use. Besides its antimalarial property, chloroquine also was used in anti-inflammatory and antivirus, especially in antitumor therapy. A mount of data showed that chloroquine mainly relied on autophagy inhibition to exert its antitumor effects. However, recently, more and more researches have revealed that chloroquine acts through other mechanisms that are autophagy-independent. Nevertheless, the current reviews lacked a comprehensive summary of the antitumor mechanism and combined pharmacotherapy of chloroquine. So here we focused on the antitumor properties of chloroquine, summarized the pharmacological mechanisms of antitumor progression of chloroquine dependent or independent of autophagy inhibition. Moreover, we also discussed the side effects and possible application developments of chloroquine. This review provided a more systematic and cutting-edge knowledge involved in the anti-tumor mechanisms and combined pharmacotherapy of chloroquine in hope of carrying out more in-depth exploration of chloroquine and obtaining more clinical applications.


Assuntos
Antineoplásicos , Autofagia , Cloroquina , Neoplasias , Cloroquina/farmacologia , Cloroquina/uso terapêutico , Humanos , Neoplasias/tratamento farmacológico , Autofagia/efeitos dos fármacos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Antineoplásicos/química , Animais , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico
20.
World J Gastroenterol ; 30(24): 3036-3043, 2024 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-38983959

RESUMO

Autophagy, a conserved cellular degradation process, is crucial for various cellular processes such as immune responses, inflammation, metabolic and oxidative stress adaptation, cell proliferation, development, and tissue repair and remodeling. Dysregulation of autophagy is suspected in numerous diseases, including cancer, neurodegenerative diseases, digestive disorders, metabolic syndromes, and infectious and inflammatory diseases. If autophagy is disrupted, for example, this can have serious consequences and lead to chronic inflammation and tissue damage, as occurs in diseases such as Chron's disease and ulcerative colitis. On the other hand, the influence of autophagy on the development and progression of cancer is not clear. Autophagy can both suppress and promote the progression and metastasis of cancer at various stages. From inflammatory bowel diseases to gastrointestinal cancer, researchers are discovering the intricate role of autophagy in maintaining gut health and its potential as a therapeutic target. Researchers should carefully consider the nature and progression of diseases such as cancer when trying to determine whether inhibiting or stimulating autophagy is likely to be beneficial. Multidisciplinary approaches that combine cutting-edge research with clinical expertise are key to unlocking the full therapeutic potential of autophagy in digestive diseases.


Assuntos
Autofagia , Doenças do Sistema Digestório , Humanos , Autofagia/efeitos dos fármacos , Doenças do Sistema Digestório/metabolismo , Doenças do Sistema Digestório/patologia , Doenças do Sistema Digestório/terapia , Doenças do Sistema Digestório/fisiopatologia , Doenças do Sistema Digestório/imunologia , Animais , Progressão da Doença
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