Expression Of LC3B Research Articles

Protective effects of pulsed electromagnetic field therapy attenuates autophagy and apoptosis in osteoporotic osteoarthritis model rats by activating PPARγ

ABSTRACT Osteoporotic osteoarthritis (OPOA) is a specific phenotype of OA with high incidence and severe cartilage damage. This study aimed to explore the protective efficacy of PEMF on the progression of OPOA and observed the effects of PEMF on PPARγ, autophagy- and apoptosis-related proteins in OPOA rats. Rats were randomly divided into three groups: control group, OPOA group, and PEMF group (n = 6). One week after surgery, the rats in PEMF group were subjected to PEMF (3.82 mT, 8 Hz, 40 min/day and 5 day/week) for 12 weeks. Results showed that PEMF retarded cartilage degeneration and bone loss, as evidenced by pathological staining image, decreased MMP-13 expression and increased bone mineral density. PEMF inhibited the serum levels of inflammatory cytokines, and the expressions of caspase-3 and caspase-8, while upregulated the expression of PPARγ. Moreover, PEMF significantly improved the autophagy disorders, represented by decrease expressions of Beclin-1, P62, and LC3B. The research demonstrates that PEMF can effectively prevent cartilage and subchondral bone destruction in OPOA rats. The potential mechanism may be related to upregulation of PPARγ, inhibition of chondrocyte apoptosis and inflammation, and improvement of autophagy disorder. PEMF therapy thus shows promising application prospects in the treatment of postmenopausal OA.

2-iodohexadecanal induces autophagy during goiter involution

BackgroundIodine plays an important role in thyroid physiology and biochemistry. The thyroid is capable of producing different iodolipids such as 2-iodohexadecanal (2-IHDA). Data from different laboratories have shown that 2-IHDA inhibits several thyroid parameters and it has been postulated as intermediary on the action of iodide function. ObjectiveTo explore different mechanisms involved during the involution of the hyperplastic thyroid gland of Wistar rats towards normality induced by 2-IHDA. Methods: Goiter was induced by the administration of MMI for 10 days, then the treatment was discontinued and Wistar rats were injected with 2-IHDA or KI. ResultsDuring involution, 2-IHDA treatment reduced PCNA expression compared to spontaneous involution. KI treatment caused an increase of Caspase-3 activity and TUNEL-positive cells. In contrast, 2-IHDA failed to alter this value but induced an increase of LC3B expression. KI but not 2-IHDA led to an increase in peroxides levels, catalase and glutathione peroxidase activity. ConclusionsWe demonstrated that 2-IHDA, in contrast to iodide, did not lead to an increase in oxidative stress or apoptosis induction, indicating that the involution triggered by 2-IHDA in Wistar rats, is primarily due to the inhibition of cell proliferation and the induction of autophagy.

Sodium fluoride-induced autophagy of ameloblast-like cells via the p-ULk1/ATG13/LC3B pathway invitro.

To investigate the effects of sodium fluoride on the ameloblast and reveal the mechanism of dental fluorosis. Mouse ameloblast-like cell line (ALC) cells were treated with various concentrations of NaF, and subjected to Incucyte, fluorescence immunoassay, transmission electron microscopy, reverse transcription quantitative polymerase chain reaction (RT-qPCR), western blot for autophagy examination, alkaline phosphatase and alizarin red staining for mineralization after osteogenic induction. NaF exerts a dose-dependent inhibitory effect on ALC cell growth. TEM and fluorescence immunoassay showed that 1.5 mM or higher concentrations of NaF could induce a fusion of lysosome and mitochondria, finally increasing the number of autophagosome. RT-qPCR and western blot showed that the upregulation of autophagy related gene 13 (ATG13), downregulation of phosphorylated Unc-51-like kinase 1 (p-ULK1) were found in NaF-induced autophagy of ALC cells. The knockdown of ATG13 could rescue it as well as the expression of p-ULK1 and LC3B. Besides, alizarin red staining showed that fluoride under these concentrations could promote the mineralization of ALC. The data show that fluoride in higher concentration can induce autophagy via the p-ULk1/ATG13/LC3B pathway of ALCs than lower ones promote mineralization invitro, which provides insight into the function of NaF in the autophagy and mineralization of ameloblast.

Theacrine enhances autophagy and inhibits inflammation via regulating SIRT3/FOXO3a/Parkin pathway.

Psoriasis, a common chronic inflammatory skin condition, impacts around 2%-3% of the global population. Theacrine is recognized for its potential anti-inflammatory and antioxidant properties. However, the role of theacrine in psoriasis remains unclear. To investigate the effects of theacrine on psoriasis and explore the underlying signaling pathways. For imiquimod (IMQ)-induced Psoriasis-like mice, the psoriatic inflammation was monitored using Psoriasis Area and Severity Index (PASI). The skin damage was observed using Hematoxylin and Eosin staining. The KI67 and CD4 in skin tissues were assessed using Immunohistochemistry analysis. Western blots were performed to evaluate the expression of Keratin 1 (KRT1), KRT6, LC3, P62, Beclin1, T-bet, GATA3, RAR-related orphan receptor (ROR)-γt, Sirtuin-3 (SIRT3), Forkhead Box O3a (FOXO3a) and Parkin. Additionally, LC3B expression was analyzed using an immunofluorescent assay, while flow cytometry was performed to analyze the percentage of Th17, Th1, and Th2 positive cells in skin-draining lymph node. Theacrine improved skin condition by reducing hyperkeratosis and acanthosis, lowering PASI scores, and decreasing KI67-positive cells. Theacrine also modulated keratin expression, elevating KRT1 while reducing KRT6 levels. Theacrine enhanced autophagy indicated by an increased LC3-II/LC3-I ratio and Beclin1, while reduced P62 levels. Additionally, Theacrine reduced CD4-positive cells and suppressed Th17 and Th1 cell activation. Theacrine activated the FOXO3a/Parkin pathway by upregulating SIRT3 expression, and down-regulation of SIRT3 counteracted theacrine's effects in psoriasis-like mice. Theacrine inhibits skin damage, promotes autophagy, and mediates inflammation in IMQ-induced psoriasis mice via upregulating SIRT3 to activate FOXO3a/Parkin pathway, positioning theacrine as a candidate for psoriasis treatment.

The modulatory effect of Lactobacillus gasseri ATCC 33323 on autophagy induced by extracellular vesicles of Helicobacter pylori in gastric epithelial cells in vitro

Helicobacter pylori has been recognized as a true pathogen, which is associated with various gastroduodenal diseases, and gastric adenocarcinoma. The crosstalk between H. pylori virulence factors and host autophagy remains challenging. H. pylori can produce extracellular vesicles (EVs) that contribute to gastric inflammation and malignancy. Some probiotic strains have been documented to modulate cell autophagy process. This study was aimed to investigate the modulatory effect of cell-free supernatant (CFS) obtained from Lactobacillus gasseri ATCC 33323 on autophagy induced by H. pylori-derived EVs. EVs were isolated from two clinical H. pylori strains (BY-1 and OC824), and characterized using transmission electron microscopy (TEM) and dynamic light scattering (DLS). The viability of AGS cells was assessed after exposure to different concentrations of H. pylori EVs, and L. gasseri CFS. Based on MTT assay and Annexin V-FITC/PI staining, 50 μg/ml of H. pylori EVs and 10 % v/v of L. gasseri CFS were used for further cell treatment experiments. Autophagy was examined using acridin orange (AO) staining, RT-qPCR analysis for autophagy mediators (LC3B, ATG5, ATG12, ATG16L1, BECN1, MTOR, and NOD1), and western blotting for LC3B expression. H. pylori EVs were detected to range in size from 50 to 200 nm. EVs of both H. pylori strains and L. gasseri CFS showed no significant effect on cell viability as compared to untreated cells. H. pylori EVs promoted the development of acidic vesicular organelles and the expression of autophagy-related genes (LC3B, ATG5, ATG12, ATG16L1, BECN1, and NOD1), and decreased the expression of MTOR in AGS cells at 12 and 24 h time periods. In addition, the production of LC3B was increased following 12 h of treatment in AGS cells. In contrast, L. gasseri CFS effectively inhibited EVs-induced autophagy, as evidenced by reduced acidic vesicular organelle formation and modulation of autophagy markers. Our study indicated that L. gasseri CFS can effectively suppress H. pylori EV-induced autophagy in AGS cells. Further investigations are required to decipher the mechanism of action L. gasseri CFS and its metabolites on autophagy inhibition induced by H. pylori.

Fangji Huangqi decoction ameliorates membranous nephropathy through the upregulation of BNIP3-mediated mitophagy

Ethnopharmacological relevanceFangji Huangqi Decoction (FJHQ), a traditional Chinese medicinal formula outlined in Zhang Zhongjing's “Jin Gui Yao Lue” during the Han Dynasty, is often used to treat conditions characterized by symptoms like edema and dysuria, including membranous nephropathy (MN). Despite its proven clinical effectiveness, the exact mechanisms through which FJHQ acts on MN remain elusive. Aim of the studyThis study aimed to investigate whether FJHQ enhances BNIP3-mediated mitophagy in podocytes by promoting BNIP3 expression and whether this improvement leads to the amelioration of MN. Materials and methodsIn this study, by establishing passive Heymann nephritis (PHN) rats, an experimental rat model of MN induced by sheep anti-rat Fx1A serum, we evaluated the effects of FJHQ in vivo. In vitro experiments were carried out by treating primary podocytes with experimental rat serum. Furthermore, the potential mechanism by which FJHQ acts through BNIP3 was further examined by transfecting primary podocytes with the siRNA of BNIP3 or the corresponding control vector. ResultsAfter 4 weeks, significant kidney damage was observed in the rats in the model group, comparatively, FJHQ markedly decreased urine volume, 24-h urinary protein, blood urea nitrogen (BUN), creatinine (Scr), and increased serum total albumin (ALB). Histology showed that FJHQ caused significant improvements in glomerular hyperplasia, and IgG immune complex deposition in MN rats. JC-1 fluorescence labelling and flow cytometry analysis showed that FJHQ could significantly increase mitochondrial membrane potential in vivo. In the mitochondria of MN model rats, FJHQ was able to down-regulate the expression of P62 and up-regulate the expression of BNIP3, LC3B, and LC3 II/LC3 I, according to Western blot and immunofluorescence studies. Furthermore, FJHQ has been shown to significantly up-regulate mitochondrial membrane potential, down-regulate P62 expression in mitochondria, and up-regulate the expression of BNIP3, LC3B, and LC3 II/LC3 I in mitochondria at the cellular level. After the administration of the autophagy inhibitor chloroquine, the serum of rats treated with FJHQ further increased the expression of LC3 II/LC3 I in primary podocytes, showing higher autophagy flow. After the interference of BNIP3 in podocytes, the effect of FJHQ on mitochondrial membrane potential and autophagy-related proteins almost disappeared. ConclusionFJHQ enhanced mitophagy in podocytes by promoting the expression of BNIP3, thereby contributing to the amelioration of MN. This work reveals the possible underlying mechanism by which FJHQ improves MN and provides a new avenue for MN treatment.

AKR1C3 silencing inhibits autophagy-dependent glycolysis in thyroid cancer cells by inactivating ERK signaling.

Thyroid cancer is a highly differentiated and poorly malignant tumor. Interfering with glycolysis has become an effective means of controlling cancer progression and autophagy is negatively correlated with glycolysis. Aldo-keto reductase family 1 member C3 (AKR1C3) has been demonstrated to be highly expressed in thyroid cancer tissue and the higher AKR1C3 expression predicted the worse prognosis. We aimed to explore whether AKR1C3 could affect thyroid cancer progression by regulating autophagy-dependent glycolysis. AKR1C3 expression in thyroid cancer cells was detected by western blot. Then, AKR1C3 was knocked down by transfection with short hairpin RNA specific to AKR1C3 in the absence or presence of 3-methyladenine (3-MA) or PMA treatment. Cell cycle and apoptosis was detected by flow cytometry. Immunofluorescence staining was used to analyze LC3B expression. Extracellular acidification, glucose uptake and lactic acid secretion were detected. To evaluate the tumorigenicity of AKR1C3 insufficiency on thyroid cancer in vivo, TPC-1 cells with AKR1C3 knockdown were injected subcutaneously into nude mice. Then, cyclinD1 and Ki67 expression in tumorous tissues was measured by immunohistochemical analysis. Apoptosis was assessed by terminal-deoxynucleoitidyl transferase mediated nick end labeling staining. Additionally, the expression of proteins related to cell cycle, apoptosis, glycolysis, autophagy, and extracellular signal-regulated kinase (ERK) signaling in cells and tumor tissues was assessed by western blot. Highly expressed AKR1C3 was observed in thyroid cancer cells. AKR1C3 knockdown induced cell cycle arrest and apoptosis of TPC-1 cells. Besides, autophagy was activated and glycolysis was inhibited following AKR1C3 silencing, and 3-MA treatment restored the impacts of AKR1C3 silencing on glycolysis. The further experiments revealed that AKR1C3 insufficiency inhibited ERK signaling and PMA application reversed AKR1C3 silencing-induced autophagy in TPC-1 cells. The in vivo results suggested that AKR1C3 knockdown inhibited the development of subcutaneous TPC-1 tumors in nude mice and inactivated the ERK signaling. Collectively, AKR1C3 silencing inhibited autophagy-dependent glycolysis in thyroid cancer by inactivating ERK signaling.

Investigating the Association between the Autophagy Markers LC3B, SQSTM1/p62, and DRAM and Autophagy-Related Genes in Glioma.

High-grade gliomas are extremely fatal tumors, marked by severe hypoxia and therapeutic resistance. Autophagy is a cellular degradative process that can be activated by hypoxia, ultimately resulting in tumor advancement and chemo-resistance. Our study aimed to examine the link between autophagy markers' expression in low-grade gliomas (LGGs) and high-grade gliomas (HGGs). In 39 glioma cases, we assessed the protein expression of autophagy markers LC3B, SQSTM1/p62, and DRAM by immunohistochemistry (IHC) and the mRNA expression of the autophagy genes PTEN, PI3K, AKT, mTOR, ULK1, ULK2, UVRAG, Beclin 1, and VPS34 using RT-qPCR. LC3B, SQSTM1/p62, and DRAM expression were positive in 64.1%, 51.3%, and 28.2% of glioma cases, respectively. The expression of LC3B and SQSTM1/p62 was notably higher in HGGs compared to LGGs. VPS34 exhibited a significant differential expression, displaying increased fold change in HGGs compared to LGGs. Additionally, it exhibited robust positive associations with Beclin1 (rs = 0.768), UVRAG (rs = 0.802), and ULK2 (rs = 0.786) in HGGs. This underscores a potential association between autophagy and the progression of gliomas. We provide preliminary data for the functional analysis of autophagy using a cell culture model and to identify potential targets for therapeutic interventions.

Disruption of Autophagic Flux and Treatment with the PDPK1 Inhibitor GSK2334470 Synergistically Inhibit Renal Cell Carcinoma Pathogenesis.

Background: Renal cell carcinoma (RCC) frequently exhibits activating PI3K-Akt-mTOR pathway mutations. 3-Phosphoinositide-dependent kinase 1 (PDPK1 or PDK1) has been established to play a pivotal role in modulating PI3K pathway signaling. mTOR is the main autophagy-initiating factor. However, limited advances have been made in understanding the relationship between PDPK1 and autophagy in RCC. Methods: GSK2334470 (GSK470), a novel and highly specific inhibitor of PDPK1, was selected to investigate the anticancer effects in two RCC cell lines. Cell growth was assessed by CCK-8 test and colony formation. Changes in the protein levels of key Akt/mTOR pathway components and apoptosis markers were assessed by Western blotting. Autophagy was assessed by using LC3B expression, transmission electron microscopy, and a tandem mRFP-EGFP-LC3 construct. The effect of PDPK1 and autophagy inhibitor chloroquine in RCC in vivo was examined in a mouse tumor-bearing model. Results: GSK470 significantly inhibited cell proliferation and induces apoptosis in A498 and 786-O RCC cells. GSK470 downregulates the phosphorylation of PDPK1, thereby inhibiting downstream phosphorylation of Akt1 at Thr308 and Ser473 and mTOR complex 1 (mTORC1) activity. Treatment with insulin-like growth factor-1 (IGF-1) partially restored GSK470-induced behaviors/activities. Interestingly, treatment of A498 and 786-O cells with GSK470 or siPDPK1 induced significant increases in the hallmarks of autophagy, including autophagosome accumulation, autophagic flux, and LC3B expression. Importantly, GSK470 and chloroquine synergistically inhibited the growth of RCC cells in vitro and in xenograft models, supporting the protective role of autophagy activation upon blockade of the PDPK1-Akt-mTOR signaling pathway. Conclusion: Our study provides new insight into PDPK1 inhibition combined with autophagy inhibition as a useful treatment strategy for RCC.

Immunohistochemical Expression of Autophagy-Related Marker (LC3B) and Stem Cell Marker (CD44) in Molecular Subtypes of Breast Cancer.

Breast cancer (BC) is among the most prevalent aggressive type of malignancy affecting females worldwide. Despite the advance in early detection and management of BC; recurrence, metastasis and mortality remains high. This may be attributed to heterogeneity of BC which explained by the presence of breast cancer stem cells (BCSCs). BCSCs is characterized by their ability of self-renewal, unlimited proliferation and their differentiation potential. BCSCs maintain their activity through process of autophagy. Autophagy is a catabolic pathway important for maintenance of cellular hemostasis in response to different stressful conditions. Autophagy allows BCSCs to adapt to different stressful conditions. So, it protects BCSCs from cytotoxic effects of anti-cancer therapy and anticancer resistance. Formalin-fixed paraffin embedded fifty specimens of Invasive duct carcinoma of no special type(IDC/NST) of breast was selected and immunostained with stem cell marker CD44 and autophagy related marker LC3B antibodies. Correlation with different clinicopathological, histopathological characteristics and molecular subtypes of studied specimens were evaluated. Both CD44 and LC3B expression were significantly associated with lymph nodal metastasis (p =0.001 and 0.010 respectively), advanced pathological stage (p= 0.045 and 0.004 respectively) and with triple negative molecular subtype of BC (p=0.044 and 0.048 respectively). Statistically positive correlation was also found between both tumor markers expression. Results of this study suggests that CD44 and LC3B expression play a role in the clinical behavior and progression of different molecular subtypes of BC.

Fucoidan alleviated autoimmune diabetes in NOD mice by regulating pancreatic autophagy through the AMPK/mTOR1/TFEB pathway.

The present study investigated the effect and its underlying mechanisms of fucoidan on Type 1 diabetes mellitus (T1DM) in non-obese diabetic (NOD) mice. Twenty 7-week-old NOD mice were used in this study, and randomly divided into two groups (10 mice in each group): the control group and the fucoidan treatment group (600 mg/kg. body weight). The weight gain, glucose tolerance, and fasting blood glucose level in NOD mice were detected to assess the development of diabetes. The intervention lasted for 5 weeks. The proportions of Th1/Th2 cells from spleen tissues were tested to determine the anti-inflammatory effect of fucoidan. Western blot was performed to investigate the expression levels of apoptotic markers and autophagic markers. Apoptotic cell staining was visualized through TdT-mediated dUTP nick-end labeling (TUNEL). The results suggested that fucoidan ameliorated T1DM, as evidenced by increased body weight and improved glycemic control of NOD mice. Fucoidan down-regulated the Th1/Th2 cells ratio and decreased Th1 type pro-inflammatory cytokines' level. Fucoidan enhanced the mitochondrial autophagy level of pancreatic cells and increased the expressions of Beclin-1 and LC3B II/LC3B I. The expression of p-AMPK was up-regulated and p-mTOR1 was inhibited, which promoted the nucleation of transcription factor EB (TFEB), leading to autophagy. Moreover, fucoidan induced apoptosis of pancreatic tissue cells. The levels of cleaved caspase-9, cleaved caspase-3, and Bax were up-regulated after fucoidan treatment. Fucoidan could maintain pancreatic homeostasis and restore immune disorder through enhancing autophagy via the AMPK/mTOR1/TFEB pathway in pancreatic cells.

Naringin Mediated Silver Nanoparticles Attenuate Rheumatoid Arthritis in Mice by Modulation of Autophagy and PI3K/AKT/mTOR Signaling Pathway

Background: Rheumatoid arthritis (RA) is characterized as an autoimmune inflammatory disease associated with synovial joints. Naringin (NG) is a flavanone type of glycoside found in grapes and citrus fruits, and reported to be active against RA. However, poor bioavailability and low solubility restricted its clinical use. Therefore, considering the issue, the present study was focused on the development, characterization and exploration of the protective efficacy of naringin-mediated silver nanoparticles (AgNNPs) against RA by using in vitro and in vivo methods. Methods: AgNNPs were biosynthesized by co-precipitation approach and characterized by different techniques. Biofabricated AgNNPs were employed to analyze the anti-arthritic effects by using an in vivo mouse model, i.e., type II collagen-induced arthritis (CIA) model and various parameters including degree of paw swelling, polyarthritis index score as well as level of cytokines in serum were evaluated. The effect of AgNNPs on the proliferation level of fibroblast-like synoviocytes (FLS) induced by tumor necrosis factor-[Formula: see text](TNF-[Formula: see text]) was also taken into account. Western blot and immunofluorescence staining were employed to detect the status of autophagy and underlying PI3K/AKT/mTOR pathway in the CIA model and FLSs. Results: Various characterization techniques confirmed the formation of spherical crystalline nanoparticles with a size range of 40-80 nm. In vivo study results revealed that AgNNPs significantly reduced (P < 0.01) the RA-associated damaging effects such as level of paw edema, and pro-inflammatory cytokines along with inhibition of FLSs proliferation associated with TNF-[Formula: see text]. Level of autophagosome and protein expression of LC3B in TNF-[Formula: see text]induced FLSs, LC3-II and Beclin-1 were enhanced whereas mTOR and AKT phosphorylation were reduced by AgNNPs treatment. Conclusion: The study exhibited an ameliorative effect of biosynthesized AgNNPs against RA by modulating autophagy through PI3K/AKT/mTOR signaling pathway.

NUPR1 contributes to activate TFE3-dependent autophagy leading to cervical cancer proliferation

Cervical cancer is a malignant tumor that occurs in the cervix of women and endangers their lives. In this study, we aimed to assess the roles of NUPR1 and TFE3 in cervical cancer. The Cancer Genome Atlas (TCGA) database was used to assess the correlation between NUPR1 and TFE3 expression in cervical cancer. By silencing NUPR1 and TFE3, and through 3-MA treatment, we determined whether their silencing could lead to lysosomal dysfunction, thereby inhibiting autophagy and cervical cancer cell proliferation. Their roles were further analyzed using molecular biological methods. Silencing NUPR1 and TFE3 inhibited cell proliferation and decreased the expression levels of autophagy-related genes, p62 and LC3B. By tracing lysosomes within cells, NUPR1 and TFE3 knockdown were found to induce lysosomal dysfunction, thereby inhibiting autophagy. In vivo experimental studies have shown that knockdown of NUPR1 and TFE3 can inhibit tumor growth, while reducing the ki67, p62, and LC3B expression levels and promoting apoptosis. Furthermore, the expression levels of lamp1 and lamp2, and the phosphorylation of PI3K (p-PI3K) and Akt (p-Akt) were significantly reduced after NUPR1 and TFE3 knockdown. However, treatment with 3-MA and overexpression of TFE3 could partially reverse the effect of silencing NUPR1. Overall, silencing NUPR1 reduced autophagy by inhibiting TFE3 in cervical cancer. Our results supply new evidence for the use of NUPR1 as a therapeutic target in cervical cancer.

Antiproliferative Activity of Gibbosic Acid H through Induction of G0/G1 Cell Cycle Arrest and Apoptosis in Human Lung Cancer Cells.

Lung cancer is one of the most common causative cancers worldwide. Particularly, non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer cases. NSCLC is a serious form of lung cancer that requires prompt diagnosis, and the 5-year survival rate for patients with this disease is only 24%. Gibbosic acid H (GaH), a natural lanostanoid obtained from the Ganoderma species (Ganodermataceae), has antiproliferative activities against colon and lung cancer cells. The aim of the present study was to evaluate the antiproliferative activity of GaH in NSCLC cells and to elucidate the underlying molecular mechanisms. GaH was found to induce G0/G1 cell cycle arrest and autophagy by activating adenosine monophosphate-activated protein kinase in A549 and H1299 cells. The induction of this cell cycle arrest was associated with the downregulation of cyclin E1 and CDK2. Additionally, the induction of autophagy by GaH was correlated with the upregulation of LC3B, beclin-1, and p53 expression. GaH also induced apoptosis by upregulating cleaved caspase-3 and Bax in the lung cancer cells. These findings suggest that GaH has a potential in the growth inhibition of human lung cancer cells.

The HIF-1/ BNIP3 pathway mediates mitophagy to inhibit the pyroptosis of fibroblast-like synoviocytes in rheumatoid arthritis

BackgroundSynovial hypoxia, a critical pathological characteristic of rheumatoid arthritis (RA), significantly contributes to synovitis and synovial hyperplasia. In response to hypoxic conditions, fibroblast-like synoviocytes (FLS) undergo adaptive changes involving gene expression modulation, with hypoxia-inducible factors (HIF) playing a pivotal role. The regulation of BCL2/adenovirus e1B 19 kDa protein interacting protein 3 (BNIP3) and nucleotide-binding oligomerization segment-like receptor family 3 (NLRP3) expression has been demonstrated to be regulated by HIF-1. The objective of this study was to examine the molecular mechanism that contributes to the aberrant activation of FLS in response to hypoxia. Specifically, the interaction between BNIP3-mediated mitophagy and NLRP3-mediated pyroptosis was conjointly highlighted. MethodsThe research methodology employed Western blot and immunohistochemistry techniques to identify the occurrence of mitophagy in synovial tissue affected by RA. Additionally, the levels of mitophagy under hypoxic conditions were assessed using Western blot, immunofluorescence, quantitative polymerase chain reaction (qPCR), and CUT&Tag assays. Pyroptosis was observed through electron microscopy, fluorescence microscopy, and Western blot analysis. Furthermore, the quantity of reactive oxygen species (ROS) was measured. The silencing of HIF-1α and BNIP3 was achieved through the transfection of short hairpin RNA (shRNA) into cells. ResultsIn the present study, a noteworthy increase in the expression of BNIP3 and LC3B was observed in the synovial tissue of patients with RA. Upon exposure to hypoxia, FLS of RA exhibited BNIP3-mediated mitophagy and NLRP3 inflammasome-mediated pyroptosis. It appears that hypoxia regulates the expression of BNIP3 and NLRP3 through the transcription factor HIF-1. Additionally, the activation of mitophagy has been observed to effectively inhibit hypoxia-induced pyroptosis by reducing the intracellular levels of ROS. Conclusion: In summary, the activation of FLS in RA patients under hypoxic conditions involves both BNIP3-mediated mitophagy and NLRP3 inflammasome-mediated pyroptosis. Additionally, mitophagy can suppress hypoxia-induced FLS pyroptosis by eliminating ROS and inhibiting the HIF-1α/NLRP3 pathway.

M2 Muscarinic Receptor Stimulation Induces Autophagy in Human Glioblastoma Cancer Stem Cells via mTOR Complex-1 Inhibition.

Although autophagy is a pro-survival process of tumor cells, it can stimulate cell death in particular conditions and when differently regulated by specific signals. We previously demonstrated that the selective stimulation of the M2 muscarinic receptor subtype (mAChR) negatively controls cell proliferation and survival and causes oxidative stress and cytotoxic and genotoxic effects in both GBM cell lines and GBM stem cells (GSCs). In this work, we have evaluated whether autophagy was induced as a downstream mechanism of the observed cytotoxic processes induced by M2 mAChR activation by the orthosteric agonist APE or the dualsteric agonist N8-Iper (N8). To assess the activation of autophagy, we analyzed the expression of LC3B using Western blot analysis and in LC3B-EGFP transfected cell lines. Apoptosis was assessed by measuring the protein expression of Caspases 3 and 9. Our data indicate that activation of M2 mAChR by N8 promotes autophagy in both U251 and GB7 cell lines as suggested by the LC3B-II expression level and analysis of the transfected cells by fluorescence microscopy. Autophagy induction by M2 mAChRs is regulated by the decreased activity of the PI3K/AKT/mTORC1 pathway and upregulated by pAMPK expression. Downstream of autophagy activation, an increase in apoptosis was also observed in both cell lines after treatment with the two M2 agonists. N8 treatment causes autophagy via pAMPK upregulation, followed by apoptosis in both investigated cell lines. In contrast, the absence of autophagy in APE-treated GSC cells seems to indicate that cell death could be triggered by mechanisms alternative to those observed for N8.

A novel ruthenium complex with 5-fluorouracil suppresses colorectal cancer stem cells by inhibiting Akt/mTOR signaling

[Ru(5-FU)(PPh3)2(bipy)]PF6 (Ru/5-FU) is a novel ruthenium complex with 5-fluorouracil with promising potential against colorectal cancer (CRC). In the present study, we investigated the molecular mechanism of Ru/5-FU action in HCT116 CRC cells. Ru/5-FU exhibited potent cytotoxicity on a panel of cancer cell lines and on primary cancer cells and induced apoptosis in HCT116 CRC cells. Ru/5-FU reduced AKT1 gene transcripts, as well as the expression of Akt1 and Akt (pS473) and downstream Akt proteins mTOR (pS2448), S6 (pS235/pS236), 4EBP1 (pT36/pT45), GSK-3β (pS9) and NF-κB p65 (pS529), but not Akt upstream proteins Hsp90 and PI3K p85/p55 (pT458/pT199), indicating an inhibitory action of Akt/mTOR signaling. Ru/5-FU increased LC3B expression and reduced p62/SQSTM1 levels, indicating autophagy induction. Curiously, the autophagy inhibitors 3-methyladenine and chloroquine increased Ru/5-FU-induced cell death, indicating an induction of cytoprotective autophagy by this compound. Ru/5-FU also reduced clonogenic survival, as well as the percentage of CD133+ cells and colonosphere formation, indicating that Ru/5-FU can suppress stem cells in HCT116 cells. Ru/5-FU inhibited cell migration and invasion in wound healing assays and Transwell cell invasion assays, along with a reduction in vimentin expression and an increase in E-cadherin levels, indicating that Ru/5-FU can interfere with epithelial-mesenchymal transition. Ru/5-FU also inhibited in vivo HCT116 cell development and experimental lung metastases in mouse xenograft models. Altogether, these results indicate that Ru/5-FU is an anti-CRC chemotherapy drug candidate with the ability to suppress stemness in CRC cells by inhibiting Akt/mTOR signaling.

Investigation of the effects of pulmonary adenomatosis on oxidative dna damage, inflammation, apoptosis, and autophagy in lung tissues in sheep

Pulmonary adenomatosis infections are an infectious and neoplastic disease that affects the respiratory system in sheep. Although the existence and prevalence of this disease have been revealed in studies, there is no definitive treatment today. Diagnosis of the disease; It can be detected by clinical, macroscopic, microscopic, and immunohistochemical methods. Although there are studies reporting the existence and prevalence of the disease today, the pathogenesis has not been fully elucidated. In this study, the effects of pulmonary adenomatosis (PA) infections on lung tissue oxidative DNA damage, inflammation, apoptosis, and autophagy were investigated in sheep. Care was taken to ensure that the lung samples used as negative controls did not show any macroscopic signs of pneumonia. The presence of tumoral foci was examined in the lung tissues to be used as positive control. Then the samples were examined histopathologically. All samples obtained were examined immunohistochemically for the presence of Jaagsiekte Sheep Retrovirus (JSRV) antibodies. In the study, the expression levels of IL1-β, Tnf-α, 8-OHdG, Bcl-2, BAX, Caspase3, LC3B, and LC3A were examined in 6 negative and 12 positive lung tissue samples for PA. In the lung tissues, proliferation of bronchiolar and alveolar epithelial cells, as well as alveolar macrophages and neutrophil leukocyte cell infiltration in their lumens, were observed. It revealed that severe OPA cases had more severe levels of IL1-β, Tnf-α, 8-OHdG, BAX, Caspase3, LC3A, and LC3B expressions than mild cases did. As a result of the research, in cases where the severity of tumor foci due to PA in lung tissues increased; Oxidative stress, inflammation, apoptosis, and autophagy were found to be significantly increased.

Enhancer-mediated FOXO3 expression promotes MSC adipogenic differentiation by activating autophagy

BackgroundMesenchymal stem cells (MSCs) are pluripotent stem cells capable of differentiating into osteocytes, adipocytes and chondrocytes. However, in osteoporosis, the balance of differentiation is tipped toward adipogenesis and the key mechanism is controversial. Researches have shown that, as upstream regulatory elements of gene expression, enhancers ar involved in the expression of identity genes. In this study, we identified enhancers-mediated gene FOXO3 promoting MSC adipogenic differentiation by activating autophagy. MethodsWe integrated data of RNA sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-seq) and ATAC-sequencing (ATAC-seq) to find the identity gene FOXO3. The expression of FOXO3 protein, adipogenic transcription factors and the substrate of autophagy were measured by western blotting. The Oil Red O (ORO) staining was used to visualize the adipogenesis of MSCs. Immunohistochemistry was used to visualize the FOXO3 expression in adipocytes in bone marrow. Immunofluorescence was used to detect the expression of PPARγ and LC3B. ResultsDuring adipogenesis, enhancers redistribute to genes associated with adipogenic differentiation, among which we identified the pivotal identity gene FOXO3. FOXO3 could promote the expression of the adipogenic transcription factors PPARγ, CEBPα, and CEBPβ during adipogenic differentiation, while PPARγ, CEBPα, and CEBPβ could in turn bind to FOXO3 and continue to promote FOXO3 expression to form a positive feedback loop. Consistently elevated FOXO3 expression promotes autophagy by activating the PI3K-AKT pathway which mediates adipogenic differentiation. ConclusionsPivotal identity gene FOXO3 promotes autophagy by activating PI3K-AKT pathway, which provokes adipogenic differentiation of MSCs. Enhancer-regulated adipogenic identity gene FOXO3 could be an attractive treatment for osteoporosis.

MicroRNA Let-7a association with glycolysis-induced autophagy in locally advanced gastric cancer: Their role in prognosis and FLOT chemotherapy resistance

Locally advanced gastric cancer (LAGC) still poses a clinical challenge despite multimodality treatment due to multidrug resistance (MDR). Recently, research suggested that autophagy and metabolic regulation may be potential anticancer targets due to their crucial roles in MDR. Let-7a participates in glycolytic and autophagic regulations which are both essential for tumor progression and resistance to therapy. This study used IHC stains; GLUT4 and LC3B to evaluate glycolysis and autophagy respectively. Moreover, mRNA Let-7a was detected by quantitative reverse transcription PCR (q-PCR) in 53 cases of LAGC. Elevated glycolysis and autophagy in LAGC tissue specimens as indicated by high GLUT4 and LC3B expression were significantly associated with adverse prognostic factors such as high pathological grade, positive nodal metastasis, and advanced T stage. Lower Let-7a levels were significantly associated with high tumor grade and advanced T stage. A significant positive correlation between GLUT4 and LC3B expression was detected. Significant inverse correlations between let7a level and IHC expression of both GLUT4 and LC3B were found. Elevated glycolysis and autophagy were significantly associated with poor overall survival (OS). Furthermore, low levels of let-7a were significantly associated with poor OS compared to high levels. Glycolysis and autophagy in LAGC were significantly associated with poor FLOT chemotherapy response. Let7a mRNA relative expression was significantly decreased in cases showing post therapy partial response and sustained disease. Multivariate analysis showed that histologic tumor type, high GLUT4 and high LC3B expression were independent factors associated with poor OS. Poor survival and post FLOT chemotherapy resistance in LAGC cases were significantly related to elevated glycolysis, elevated autophagy, and reduced Let-7a expression. Accordingly, combined therapeutic targeting of these pathways could enhance chemosensitivity in LAGC.