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Frontiers in Cellular and Infection... 2024Parasites possess remarkable abilities to evade and manipulate the immune response of their hosts. is a parasitic tapeworm that causes cystic echinococcosis in animals...
Parasites possess remarkable abilities to evade and manipulate the immune response of their hosts. is a parasitic tapeworm that causes cystic echinococcosis in animals and humans. The hydatid fluid released by the parasite is known to contain various immunomodulatory components that manipulate host´s defense mechanism. In this study, we focused on understanding the effect of hydatid fluid on dendritic cells and its impact on autophagy induction and subsequent T cell responses. Initially, we observed a marked downregulation of two C-type lectin receptors in the cell membrane, CLEC9A and CD205 and an increase in lysosomal activity, suggesting an active cellular response to hydatid fluid. Subsequently, we visualized ultrastructural changes in stimulated dendritic cells, revealing the presence of macroautophagy, characterized by the formation of autophagosomes, phagophores, and phagolysosomes in the cell cytoplasm. To further elucidate the underlying molecular mechanisms involved in hydatid fluid-induced autophagy, we analyzed the expression of autophagy-related genes in stimulated dendritic cells. Our results demonstrated a significant upregulation of and , indicating the induction of autophagy machinery in response to hydatid fluid exposure. Additionally, using confocal microscopy, we observed an accumulation of LC3 in dendritic cell autophagosomes, confirming the activation of this catabolic pathway associated with antigen presentation. Finally, to evaluate the functional consequences of hydatid fluid-induced autophagy in DCs, we evaluated cytokine transcription in the splenocytes. Remarkably, a robust polyfunctional T cell response, with inhibition of Th2 profile, is characterized by an increase in the expression of and genes. These findings suggest that hydatid fluid-induced autophagy in dendritic cells plays a crucial role in shaping the subsequent T cell responses, which is important for a better understanding of host-parasite interactions in cystic echinococcosis.
Topics: Dendritic Cells; Animals; Echinococcus granulosus; Autophagy; Echinococcosis; T-Lymphocytes; Mice; Lectins, C-Type; Cytokines; Female; Autophagosomes
PubMed: 38817444
DOI: 10.3389/fcimb.2024.1334211 -
Advances in Medical Sciences May 2024Epidural analgesia has emerged as a commonly used method for relieving labor pain. However, epidural-related maternal fever (ERMF) is characterized by a high occurrence...
PURPOSE
Epidural analgesia has emerged as a commonly used method for relieving labor pain. However, epidural-related maternal fever (ERMF) is characterized by a high occurrence rate and can have detrimental consequences for the well-being of both the mother and the fetus. This study aimed to investigate the functional role and underlying mechanism of dexmedetomidine (DEX) in ERMF.
MATERIALS AND METHODS
Ropivacaine (ROP)-induced human umbilical vein endothelial cells (HUVECs) were treated with DEX and/or transfected with ALKBH5 or FUNDC1 overexpression plasmid. qPCR and Western blot were adopted for mitophagy and pyroptosis marker protein detection. Autophagosomes were observed through electron microscopy, Caspase-1/PI double-positive cells were determined using flow cytometry. Inflammation-related factors were quantified using ELISA. The N-methyladenosine (mA) modification of FUNDC1 mRNA was examined using methylated RNA immunoprecipitation (MeRIP) and the binding between ALKBH5 and FUNDC1 mRNA was confirmed by RNA immunoprecipitation (RIP).
RESULTS
In ROP-induced HUVECs, there was a significant upregulation in ALKBH5 and FUNDC1, resulting in a notable increase in inflammation, pyroptosis, and mitophagy. The administration of DEX demonstrated the ability to alleviate ROP-induced pyroptosis and promote protective mitophagy. Interestingly, DEX treatment significantly reduced the interaction between ALKBH5 and FUNDC1 mRNA, while simultaneously increasing the mA level of FUNDC1 mRNA in ROP-treated cells. Moreover, the overexpression of FUNDC1 partially reversed the effects of ALKBH5 overexpression on mitophagy and pyroptosis in HUVECs.
CONCLUSIONS
DEX can promote mitophagy and inhibit pyroptosis through the ALKBH5/FUNDC1 axis in ERMF, indicating its potential as a therapeutic strategy for clinical ERMF treatment.
PubMed: 38815927
DOI: 10.1016/j.advms.2024.05.002 -
The Journal of Biological Chemistry May 2024Extracellular secretion is an essential mechanism for α-synuclein (α-syn) proteostasis. Although it has been reported that neuronal activity affects α-syn secretion,...
Extracellular secretion is an essential mechanism for α-synuclein (α-syn) proteostasis. Although it has been reported that neuronal activity affects α-syn secretion, the underlying mechanisms remain unclear. Here, we investigated the autophagic processes that regulate the physiological release of α-syn in mouse primary cortical neurons and SH-SY5Y cells. Stimulating neuronal activity with glutamate or depolarization with high KCl enhanced α-syn secretion. This glutamate-induced α-syn secretion was blocked by a mixture of NMDA receptor antagonist AP5 and AMPA receptor antagonist NBQX, as well as by cytosolic Ca chelator BAPTA-AM. Additionally, mTOR inhibitor rapamycin increased α-syn and p62/SQSTM1 (p62) secretion, and this effect of rapamycin was reduced in primary cortical neurons deficient in the autophagy regulator beclin 1 (derived from BECN1 mice). Glutamate-induced α-syn and p62 secretion was suppressed by the knockdown of ATG5, which is required for autophagosome formation. Glutamate increased LC3-II generation and decreased intracellular p62 levels, and the increase in LC3-II levels was blocked by BAPTA-AM. Moreover, glutamate promoted co-localization of α-syn with LC3-positive puncta, but not with LAMP1-positive structures in the neuronal somas. Glutamate-induced α-syn and p62 secretion were also reduced by the knockdown of RAB8A, which is required for autophagosome fusion with the plasma membrane. Collectively, these findings suggest that stimulating neuronal activity mediates autophagic α-syn secretion in a cytosolic Ca-dependent manner, and autophagosomes may participate in autophagic secretion by functioning as α-syn carriers.
PubMed: 38815862
DOI: 10.1016/j.jbc.2024.107419 -
Frontiers in Bioscience (Landmark... May 2024Ibrutinib could increase the risk of atrial fibrillation (AF) in chronic lymphocytic leukemia (CLL) patients. However, the precise mechanism underlying ibrutinib-induced...
BACKGROUND
Ibrutinib could increase the risk of atrial fibrillation (AF) in chronic lymphocytic leukemia (CLL) patients. However, the precise mechanism underlying ibrutinib-induced AF remains incompletely elucidated.
METHODS
We investigated the proportion of ibrutinib-treated CLL patients with new-onset AF. Optical mapping was conducted to reveal the proarrhythmic effect of ibrutinib on HL-1 cells. Fluorescence staining and western blot were used to compare connexins 43 and 40 expression in ibrutinib-treated and control groups. To identify autophagy phenotypes, we used western blot to detect autophagy-related proteins, transmission electron microscopy to picture autophagosomes, and transfected mCherry-GFP-LC3 virus to label autophagosomes and lysosomes. Hydroxychloroquine as an autophagy inhibitor was administered to rescue ibrutinib-induced Cx43 and Cx40 degradation.
RESULTS
About 2.67% of patients developed atrial arrhythmias after ibrutinib administration. HL-1 cells treated with ibrutinib exhibited diminished conduction velocity and a higher incidence of reentry-like arrhythmias compared to controls. Cx43 and Cx40 expression reduced along with autophagy markers increased in HL-1 cells treated with ibrutinib. Inhibiting autophagy upregulated Cx43 and Cx40.
CONCLUSIONS
The off-target effect of ibrutinib on the PI3K-AKT-mTOR signaling pathway caused connexin degradation and atrial arrhythmia via promoting autophagy.
CLINICAL TRIAL REGISTRATION
ChiCTR2100046062, https://clin.larvol.com/trial-detail/ChiCTR2100046062.
Topics: Humans; Adenine; TOR Serine-Threonine Kinases; Autophagy; Proto-Oncogene Proteins c-akt; Piperidines; Signal Transduction; Phosphatidylinositol 3-Kinases; Connexin 43; Female; Atrial Fibrillation; Connexins; Male; Aged; Middle Aged; Gap Junction alpha-5 Protein; Arrhythmias, Cardiac
PubMed: 38812314
DOI: 10.31083/j.fbl2905201 -
Zhongguo Zhong Yao Za Zhi = Zhongguo... May 2024This study investigated the protective effect of ginsenoside Rg_1(GRg_1) on oxygen and glucose deprivation/reoxygenation(OGD/R)-injured rat adrenal...
This study investigated the protective effect of ginsenoside Rg_1(GRg_1) on oxygen and glucose deprivation/reoxygenation(OGD/R)-injured rat adrenal pheochromocytoma(PC12) cells and whether the underlying mechanism was related to the regulation of inositol-requiring enzyme 1(IRE1)-c-Jun N-terminal kinase(JNK)-C/EBP homologous protein(CHOP) signaling pathway. An OGD/R model was established in PC12 cells, and PC12 cells were randomly classified into control, model, OGD/R+GRg_1(0.1, 1, 10 μmol·L~(-1)), OGD/R+GRg_1+rapamycin(autophagy agonist), OGD/R+GRg_1+3-methyladenine(3-MA,autophagy inhibitor), OGD/R+GRg_1+tunicamycin(endoplasmic reticulum stress agonist), OGD/R+GRg_1+4-phenylbutyric acid(4-PBA, endoplasmic reticulum stress inhibitor), and OGD/R+GRg_1+3,5-dibromosalicylaldehyde(DBSA, IRE1 inhibitor) groups. Except the control group, the other groups were subjected to OGD/R treatment, i.e., oxygen and glucose deprivation for 6 h followed by reoxygenation for 6 h. Cell viability was detected by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide(MTT) assay. Apoptosis was detected by Hoechst 33342 staining, and the fluorescence intensity of autophagosomes by the monodansylcadaverine(MDC) assay. Western blot was employed to determine the expression of autophagy-related proteins(Beclin1, LC3-Ⅱ, and p62) and the pathway-related proteins [IRE1, p-IRE1, JNK, p-JNK, glucose-regulated protein 78(GRP78), and CHOP]. The results showed that GRg_1 dose-dependently increased the viability of PC12 cells and down-regulated the expression of Beclin1, LC3-Ⅱ, p-IRE1, p-JNK, GRP78, and CHOP, compared with the model group. Furthermore, GRg_1 decreased the apoptosis rate and MDC fluorescence intensity and up-regulated the expression of p62 protein. Compared with the OGD/R+GRg_1(10 μmol·L~(-1)) group, OGD/R+GRg_1+rapamycin and OGD/R+GRg_1+tunicamycin groups showed increased apoptosis rate and MDC fluorescence intensity, up-regulated protein levels of Beclin1, LC3-Ⅱ, p-IRE1, p-JNK, GRP78, and CHOP, decreased relative cell survival rate, and down-regulated protein level of p62. The 3-MA, 4-PBA, and DBSA groups exerted the opposite effects. Taken together, GRg_1 may ameliorate OGD/R-induced PC12 cell injury by inhibiting autophagy via the IRE1-JNK-CHOP pathway.
Topics: Animals; Rats; PC12 Cells; Transcription Factor CHOP; Glucose; Ginsenosides; Protein Serine-Threonine Kinases; Apoptosis; Signal Transduction; Autophagy; Endoribonucleases; JNK Mitogen-Activated Protein Kinases; Oxygen; Endoplasmic Reticulum Stress; Multienzyme Complexes
PubMed: 38812175
DOI: 10.19540/j.cnki.cjcmm.20231128.405 -
Zhongguo Zhong Yao Za Zhi = Zhongguo... May 2024To explore the active substances exerting anti-tumour effect in lemon essential oil and the molecular mechanism inhibiting the proliferation of head and neck cancer...
To explore the active substances exerting anti-tumour effect in lemon essential oil and the molecular mechanism inhibiting the proliferation of head and neck cancer cells SCC15 and CAL33, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay(MTT) was utilized to identify the active component inhibiting the proliferation of head and neck cancer cells, namely citral. The IC_(50) of citral inhibiting the proliferation of head and neck cancer cells and normal cells were also determined. In addition, a 5-ethynyl-2'-deoxyuridine(EdU) staining assay was used to detect the effect of citral on the proliferation rate of head and neck cancer cells, and a colony formation assay was used to detect the effect of citral on tumor sphere formation of head and neck cancer cells in vitro. The cell cycle arrest and apoptosis induction of head and neck cancer cells by citral were evaluated by flow cytometry, and Western blot was used to detect the effect of citral on the expression levels of cell cycle-and apoptosis-related proteins in head and neck cancer cells. The findings indicated that citral could effectively inhibit the proliferation and growth of head and neck cancer cells, with anti-tumor activity, and its half inhibitory concentrations for CAL33 and SCC15 were 54.78 and 25.23 μg·mL~(-1), respectively. Furthermore, citral arrested cell cycle at G_2/M phase by down-regulating cell cycle-related proteins such as S-phase kinase associated protein 2(SKP2), C-MYC, cyclin dependent kinase 1(CDK1), and cyclin B. Moreover, citral increased the cysteinyl aspartate-specific proteinase-3(caspase-3), cysteinyl aspartate-specific proteinase-9(caspase-9), and cleaved poly ADP-ribose polymerase(PARP). It up-regulated the level of autophagy-related proteins including microtubule associated protein 1 light chain 3B(LC3B), sequestosome 1(P62/SQSTM1), autophagy effector protein Beclin1(Beclin1), and lysosome-associate membrane protein 1(LAMP1), suggesting that citral could effectively trigger cell apoptosis and cell autophagy in head and neck cancer cells. Furthermore, the dual-tagged plasmid system mCherry-GFP-LC3 was used, and it was found that citral impeded the fusion of autophagosomes and lysosomes, leading to autophagic flux blockage. Collectively, our findings reveal that the main active anti-proliferation component of lemon essential oil is citral, and this component has a significant inhibitory effect on head and neck cancer cells. Its underlying molecular mechanism is that citral induces apoptosis and autophagy by cell cycle arrest and ultimately inhibits cell proliferation.
Topics: Humans; Cell Proliferation; Acyclic Monoterpenes; Head and Neck Neoplasms; Apoptosis; Cell Line, Tumor; Oils, Volatile; Monoterpenes; Cell Cycle Checkpoints; Citrus; Plant Oils
PubMed: 38812137
DOI: 10.19540/j.cnki.cjcmm.20240115.704 -
Brazilian Journal of Medical and... 2024Adenine nucleotide translocator 4 (Ant4), an ATP/ADP transporter expressed in the early phases of spermatogenesis, plays a crucial role in male fertility. While Ant4...
Adenine nucleotide translocator 4 (Ant4), an ATP/ADP transporter expressed in the early phases of spermatogenesis, plays a crucial role in male fertility. While Ant4 loss causes early arrest of meiosis and increased apoptosis of spermatogenic cells in male mice, its other potential functions in male fertility remain unexplored. Here, we utilized Ant4 knockout mice to delineate the effects of Ant4-deficiency on male reproduction. Our observations demonstrated that Ant4-deficiency led to infertility and impaired testicular development, which was further investigated by evaluating testicular oxidative stress, autophagy, and inflammation. Specifically, the loss of Ant4 led to an imbalance of oxidation and antioxidants. Significant ultrastructural alterations were identified in the testicular tissues of Ant4-deficient mice, including swelling of mitochondria, loss of cristae, and accumulation of autophagosomes. Our results also showed that autophagic flux and AKT-AMPK-mTOR signaling pathway were affected in Ant4-deficient mice. Moreover, Ant4 loss increased the expression of pro-inflammatory factors. Overall, our findings underscored the importance of Ant4 in regulating oxidative stress, autophagy, and inflammation in testicular tissues. Taken together, these insights provided a nuanced understanding of the significance of Ant4 in testicular development.
Topics: Animals; Male; Mice, Knockout; Testis; Oxidative Stress; Mitochondrial ADP, ATP Translocases; Mice; Autophagy; Infertility, Male; Spermatogenesis; Apoptosis; Signal Transduction
PubMed: 38808891
DOI: 10.1590/1414-431X2024e13590 -
Autophagy May 2024The noncanonical ubiquitin-like conjugation cascade involving the E1 (Atg7), E2 (Atg3, Atg10), and E3 (Atg12-Atg5-Atg16 complex) enzymes is essential for incorporation...
The noncanonical ubiquitin-like conjugation cascade involving the E1 (Atg7), E2 (Atg3, Atg10), and E3 (Atg12-Atg5-Atg16 complex) enzymes is essential for incorporation of Atg8 into the growing phagophore via covalent linkage to PE. This process is an indispensable step in autophagy. Atg8 and E1-E3 enzymes are the first subset from the core autophagy protein machinery structures that were investigated in earlier studies by crystallographic analyses of globular domains. However, research over the past decade shows that many important functions in the conjugation machinery are mediated by intrinsically disordered protein regions (IDPRs) - parts of the protein that do not adopt a stable secondary or tertiary structure, which are inherently dynamic and well suited for protein-membrane interactions but are invisible in protein crystals. Here, we summarize earlier and recent findings on the autophagy conjugation machinery by focusing on the IDPRs. This summary reveals that IDPRs, originally considered dispensable, are in fact major players and a driving force in the function of the autophagy conjugation system. : AD, activation domain of Atg7; AH, amphipathic helix; AIM, Atg8-family interacting motif; CL, catalytic loop (of Atg7); CTD, C-terminal domain; FR, flexible region (of Atg3 or Atg10); GUV, giant unilammelar vesicles; HR, handle region (of Atg3); IDPR, intrinsically disordered protein region; IDPs: intrinsically disordered proteins; LIR, LC3-interacting region; NHD: N-terminal helical domain; NMR, nuclear magnetic resonance; PE, phosphatidylethanolamine; UBL, ubiquitin like.
PubMed: 38808635
DOI: 10.1080/15548627.2024.2357496 -
The Kaohsiung Journal of Medical... Jul 2024Autophagy can be classified as degradative and secretory based on distinct functions. The small GTPase proteins Rab8a and Rab37 are responsible for secretory...
Autophagy can be classified as degradative and secretory based on distinct functions. The small GTPase proteins Rab8a and Rab37 are responsible for secretory autophagy-mediated exocytosis of IL-1β, insulin, and TIMP1 (tissue inhibitor of 54 metalloproteinase 1). Other Rab family members participating in secretory autophagy are poorly understood. Herein, we identified 26 overlapped Rab proteins in purified autophagosomes of mouse pancreatic β-cell "Min-6" and human lung cancer cell "CL1-5-Q89L" with high secretory autophagy tendency by LC-MS/MS proteomics analysis. Six Rab proteins (Rab8a, Rab11b, Rab27a, Rab35, Rab37, and Rab7a) were detected in autophagosomes of four cell lines, associating them with autophagy-related vesicle trafficking. We used CL1-5-Q89L cell line model to evaluate the levels of Rab proteins colocalization with autophagy LC3 proteins and presence in purified autophagosomes. We found five Rab proteins (Rab8a, Rab11b, Rab27a, Rab35, and Rab37) are highly expressed in the autophagosome compared to the normal control by immunoblotting under active secretion conditions. However, only Rab8a, Rab35, and Rab37 showing high colocalization with LC3 protein by cofocal microscopy. Despite the discrepancy between the image and immunoblotting analysis, our data sustains the speculation that Rab8a, Rab11b, Rab27a, Rab35, and Rab37 are possibly associated with the secretory autophagy machinery. In contrast, Rab7a shows low colocalization with LC3 puncta and low level in the autophagosome, suggesting it regulates different vesicle trafficking machineries. Our findings open a new direction toward exploring the role of Rab proteins in secretory autophagy-related cargo exocytosis and identifying the cargoes and effectors regulated by specific Rab proteins.
Topics: rab GTP-Binding Proteins; Autophagy; Humans; Animals; Mice; Autophagosomes; Cell Line, Tumor; Microtubule-Associated Proteins; Proteomics; Tandem Mass Spectrometry
PubMed: 38804615
DOI: 10.1002/kjm2.12848 -
Plant Cell Reports May 2024The VaBAM3 cloned from Vitis amurensis can enhance the cold tolerance of overexpressed plants, but VaBAM3 knock out by CRISPR/Cas9 system weakened grape callus cold...
The VaBAM3 cloned from Vitis amurensis can enhance the cold tolerance of overexpressed plants, but VaBAM3 knock out by CRISPR/Cas9 system weakened grape callus cold tolerance. In grape production, extreme cold conditions can seriously threaten plant survival and fruit quality. Regulation of starch content by β-amylase (BAM, EC: 3.2.1.2) contributes to cold tolerance in plants. In this study, we cloned the VaBAM3 gene from an extremely cold-tolerant grape, Vitis amurensis, and overexpressed it in tomato and Arabidopsis plants, as well as in grape callus for functional characterization. After exposure to cold stress, leaf wilting in the VaBAM3-overexpressing tomato plants was slightly less pronounced than that in wild-type tomato plants, and these plants were characterized by a significant accumulation of autophagosomes. Additionally, the VaBAM3-overexpressing Arabidopsis plants had a higher freezing tolerance than the wild-type counterparts. Under cold stress conditions, the activities of total amylase, BAM, peroxidase, superoxide dismutase, and catalase in VaBAM3-overexpressing plants were significantly higher than those in the corresponding wild-type plants. Furthermore, sucrose, glucose, and fructose contents in these lines were similarly significantly higher, whereas starch contents were reduced in comparison to the levels in the wild-type plants. Furthermore, we detected high CBF and COR gene expression levels in cold-stressed VaBAM3-overexpressing plants. Compared with those in VaBAM3-overexpressing grape callus, the aforementioned indicators tended to change in the opposite direction in grape callus with silenced VaBAM3. Collectively, our findings indicate that heterologous overexpression of VaBAM3 enhanced cold tolerance of plants by promoting the accumulation of soluble sugars and scavenging of excessive reactive oxygen species. These findings provide a theoretical basis for the cultivation of cold-resistant grape and support creation of germplasm resources for this purpose.
Topics: Vitis; Plant Proteins; Gene Expression Regulation, Plant; Reactive Oxygen Species; Plants, Genetically Modified; Seedlings; Arabidopsis; Cold Temperature; Solanum lycopersicum; Sugars; beta-Amylase; Starch; Cold-Shock Response
PubMed: 38802546
DOI: 10.1007/s00299-024-03236-5