Autophagic Levels Research Articles

Autophagy and Exosomes: Cross-Regulated Pathways Playing Major Roles in Hepatic Stellate Cells Activation and Liver Fibrosis.

Chronic liver injury, regardless of the underlying disease, results in gradual alteration of the physiological hepatic architecture and in excessive production of extracellular matrix, eventually leading to cirrhosis Liver cellular architecture consists of different cell populations, among which hepatic stellate cells (HSCs) have been found to play a major role in the fibrotic process. Under normal conditions, HSCs serve as the main storage site for vitamin A, however, pathological stimuli lead to their transdifferentiation into myofibroblast cells, with autophagy being the key regulator of their activation, through lipophagy of their lipid droplets. Nevertheless, the role of autophagy in liver fibrosis is multifaceted, as increased autophagic levels have been associated with alleviation of the fibrotic process. In addition, it has been found that HSCs receive paracrine stimuli from neighboring cells, such as injured hepatocytes, Kupffer cells, sinusoidal endothelial cells, which promote liver fibrosis. These stimuli have been found to be transmitted via exosomes, which are incorporated by HSCs and can either be degraded through lysosomes or be secreted back into the extracellular space via fusion with the plasma membrane. Furthermore, it has been demonstrated that autophagy and exosomes may be concomitantly or reciprocally regulated, depending on the cellular conditions. Given that increased levels of autophagy are required to activate HSCs, it is important to investigate whether autophagy levels decrease at later stages of hepatic stellate cell activation, leading to increased release of exosomes and further propagation of hepatic fibrosis.

Apoptosis triggered by cytolethal distending toxin B subunit of Helicobacter hepaticus is aggravated by autophagy inhibition in mouse hepatocytes

Hepatocytes injury caused by cytolethal distending toxin (CDT) are major events during helicobacter hepaticus (H.hepaticus) infection. Recent study showed that pre-survival autophagy was promoted against CdtB subunit induced DNA damage. In the present study, we demonstrated that inflammatory cytokines IL-6, IL-1β, TNF-α, IFN-α, IFN-γ expression and STAT phosphorylation were promoted by CdtB. Besides, CdtB decreased cell viability while promote apoptosis in mouse liver (AML12) cells. Especially, apoptotic protein caspase-9, caspase-3 and PARP were activated while the ratio of Bcl-2/Bax was decreased after CdtB treatment. Moreover, apoptosis induced by CdtB was inhibited due to Erk/p38 MAPK signaling pathway suppression performed with SB203580 or U0126. Meanwhile, we found that CdtB increased autophagic marker levels accompanied by Akt/mTOR/P70S6K signaling pathway in a dose dependent manner. To assess the correlation between autophagy and apoptosis induced by H.hepaticus, chloroquine (CQ, 50 μM) was employed to inhibit autophagy. The result showed that inhibition of autophagy with CQ treatment promoted apoptosis induced by CdtB. Altogether, all these results suggest that CdtB triggers apoptosis via MAPK/Erk/p38 signaling pathway in caspase dependent manner, which was prevented by autophagy in AML12 cells. Collectively, our findings provide new insights into the virulence potential of CdtB on the molecular pathogenesis throughout H.hepaticus infection.

Cyclosporin A alleviates trophoblast apoptosis and senescence by promoting autophagy in preeclampsia.

Abnormal extravillous trophoblast (EVT) function is closely related to preeclampsia (PE) and may be caused by inadequate autophagy, apoptosis, and senescence. Cyclosporin A (CsA) is an effective immunosuppressant that has been reported to stimulate autophagy and exert benign biological effects on EVTs. Therefore, we hypothesized that CsA may display therapeutic efficacy against PE by activating autophagy. We established the nitro-l-arginine methyl ester (l-NAME)-induced preeclamptic mice model and a hypoxia-reoxygenation (H/R) model in vitro. The effects of CsA on autophagy were evaluated by western blotting (WB). The effects of CsA on apoptosis were analyzed by Hematoxylin-eosin (H&E) staining, cell apoptosis assay and WB. Senescence-associated β-galactosidase (SA-β-gal) staining, RT-qPCR and WB were used to examine the senescence level. RT-qPCR were used to detect the senescence-associated secretory phenotype (SASP) level. DCFH-DA fluorescent probe, dihydroethidium (DHE) staining and mitochondrial membrane potential (ΔΨm) were used to detect senescence-associated mitochondrial dysfunction (SAMD). CsA alleviated PE-like symptoms and reduced placental necrosis and senescence in mice injected with l-NAME. CsA ameliorated placental SASP and SAMD level induced by l-NAME. CsA also upregulated the expression of autophagic proteins in mouse placentas disrupted using l-NAME. In vitro, we found that CsA reversed H/R-induced apoptosis and senescence, as well as decreasing SASP and SAMD levels and upregulating autophagic proteins levels. Notably, 3-methyladenine (3-MA), an early phase inhibitor of autophagosome formation, abolished the protective effects of CsA against H/R. CsA may display some therapeutic effects against PE by activating autophagy in vivo and in vitro.

Stress while lacking of control induces ventral hippocampal autophagic flux hyperactivity and a depression-like behavior

BackgroundStressed animals may perform depression-like behavior insomuch as stress-provoking blood–brain barrier (BBB) disruption, central immune activation, and autophagic flux changes. This study was undertaken to assess whether adult mice having (executive) vs. lacking (yoke) of behavioral control in otherwise equivalent stress magnitude condition, may display differences in their BBB integrity, ventral hippocampal (VH) interleukin-6 (IL-6) and autophagic flux level and VH-related depression-like behavior. To further understand the causative relation of enhanced autophagic flux and stress-primed depression-like behavior, we assessed the effects of bilateral intra-VH 3-methyladenine (3-MA), an autophagic flux inhibitor, infusion in stressed mice. MethodsAdult mice used had comparable genetic background and housing condition. Executive/yoke pairs of mice received a 10-day (1 h/day) footshock stressor regimen. Throughout the regimen, the ongoing footshock was terminated immediately contingent on the executive mouse’, while irrelevant to the respective yoke mouse’ voluntary behavior, or lasting for 7 s. Each dyad's cage-mate receiving no such regimen served as no stressor controls. ResultsYoke mice displayed disrupted BBB integrity (escalated Evans blue extravasation and decreased VH ZO-1, claudin-5 expression), increases in VH autophagic flux (increased LC3II/LC3I and decreased p62) and immobility duration in forced swimming test. Most of these indices remained unaltered in executive mice. Administration of 3-MA did not affect immobility duration in control mice, while prevented the increases in immobility duration in yoke mice. Conclusions(1) stress susceptibility may be determined by their differences in stress-coping results; (2) VH autophagic flux increase plays a permissive role in priming the stressed animals susceptible to exhibit depression-like behavior.

Effects of Ultrasound Contrast Agent-Mediated Nerve Growth Factor on Apoptosis of Retinal Ganglion Cells in Mice with Glaucoma.

With an increasing incidence in recent years, glaucoma (GL) has gradually become a global public health problem for humans of all ages. Nerve growth factor (NGF) eye drops, with well-documented stable effect in the treatment of GL, can be potentiated by the administration of NGF drugs via ultrasound contrast agent (UCA). This study analyzed the efficacy of NGF+UCA on GL mice and the influencing mechanism on retinal ganglion cells and further explored the pathological changes of GL mice under different UCA irradiation duration. In this study, we established GL mouse models and treated the mouse with NGF+UCA. The effect of NGF+UCA on intraocular pressure in mice was observed; the flash visual evoked potential of mice was compared; the changes of retinal structure, inflammation index, and oxidative stress index were observed, and autophagic protein levels were tested. Finally, the influence of UCA irradiation duration on GL symptoms was observed. The results showed that the intraocular pressure of mice decreased greatly, while their flash visual evoked potential and nervous layer of retina increased, and their ganglion cells showed stronger proliferation activity and weaker apoptosis and autophagy, indicating that UCA-mediated NGF can strongly improve the pathological condition of GL mice. In addition, PI3K/AKT pathway-associated proteins were inhibited in retina under the intervention of NGF+UCA, which further suggests that the influence of UCA-mediated NGF on GL is achieved by inhibiting autophagy of retinal ganglion cells and enhancing their apoptosis via the PI3K/AKT signaling pathway. Moreover, we found that in the treatment of GL, three weeks of UCA irradiation and six weeks caused no significant difference in the pathological manifestations and ganglion cells of mice, while after six weeks of irradiation, the level of NLRP3 in mice increased. In conclusion, UCA-mediated NGF can significantly improve the pathological condition of GL mice and improve the apoptosis of retinal ganglion cells by inhibiting autophagy, which is associated with the inhibition of the PI3K/AKT signal pathway. In terms of selection of UCA irradiation duration, three weeks of irradiation is enough to yield good clinical results.

The Link between Circadian Clock Genes and Autophagy in Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD), a progressive respiratory disease, is characterized by the alveolar epithelium injury and persistent airway inflammation. It is documented that oscillation and dysregulated expression of circadian clock genes, like Bmal1, Per1, and Per2, involved in COPD pathogenies, including chronic inflammation and imbalanced autophagy level, and targeting the associations of circadian rhythm and autophagy is promising strategies in the management and treatment of COPD. Herein, we reviewed the mechanisms of the circadian clock and the unbalance of the autophagic level in COPD, as well as the link between the two, so as to provide further theoretical bases for the study on the pathogenesis of COPD.

Size-dependent effects of polystyrene nanoplastics on autophagy response in human umbilical vein endothelial cells

Ubiquitous nanoplastics (NPs) increase exposure risks to humans through the food chain and/or other ways. However, huge knowledge gaps exist regarding the fate and adverse impact of NPs on the human cardiovascular system. Autophagy is an important catabolic pathway that disposes of cytoplasmic waste through the lysosomes. In this study, we pursued to determine the interaction and autophagy effect of polystyrene nanoplastics (PS-NPs) (100 and 500 nm in size) on human umbilical vein endothelial cells (HUVECs). The results showed both sizes of PS-NPs interacted with almost all the treated HUVECs in a time- and concentration-dependent manner, and 500 nm PS-NPs were only bound to the surface of cell membranes, whereas 100 nm PS-NPs were taken up by HUVECs and aggregated in the cytoplasm. Furthermore, exposure to 25 μg/mL of 500 nm PS-NPs for 48 h significantly increased lactate dehydrogenase release from HUVECs, while internalized 100 nm PS-NPs not only caused cell membrane damage, but also induced autophagy initiation and autophagosome formation. By a mCherry-GFP-LC3 lentivirus infection assay, we also demonstrated that autophagic flux level was impaired in response to 100 nm PS-NPs. Herein, our results provide new insight into the size-dependent internalization and autophagy response to PS-NPs in HUVECs.

Ca2+ transfer via the ER-mitochondria tethering complex in neuronal cells contribute to cadmium-induced autophagy.

Mitochondrial-associated endoplasmic reticulum (ER) membranes (MAMs) play a key role in several physiological functions, including calcium ion (Ca2+) transfer and autophagy; however, the molecular mechanism controlling this interaction in cadmium (Cd)-induced neurotoxicity is unknown. This study shows that Cd induces alterations in MAMs and mitochondrial Ca2+ levels in PC12 cells and primary neurons. Ablation or silencing of mitofusin 2 (Mfn2) in PC12 cells or primary neurons blocks the colocalization of ER and mitochondria while reducing the efficiency of mitochondrial Ca2+ uptake. Moreover, Mfn2 defects reduce interactions or colocalization between GRP75 and VDAC1. Interestingly, the enhancement of autophagic protein levels, colocalization of LC3 and Lamp2, and GFP-LC3 puncta induced by Cd decreased in Mfn2-/- or Grp75-/- PC12 cells and Mfn2- or Grp75-silenced primary neurons. Notably, the specific Ca2+ uniporter inhibitor RuR blocked both mitochondrial Ca2+ uptake and autophagy induced by Cd. Finally, this study proves that the mechanism by which IP3R-Grp75-VDAC1 tethers in MAMs is associated with the regulation of autophagy by Mfn2 and involves their role in mediating mitochondrial Ca2+ uptake from ER stores. These results give new evidence into the organelle metabolic process by demonstrating that Ca2+ transport between ER-mitochondria is important in autophagosome formation in Cd-induced neurodegeneration.

A Study on Mesoporous Silica Loaded With Novel Photosensitizers HCE6 and Oxaliplatin for the Treatment of Cholangiocarcinoma.

PurposeCholangiocarcinoma (CCA) is a malignant tumor with a high incidence. The therapeutic effect of conventional chemotherapy and radiotherapy is not obvious. Photodynamic therapy (PDT) is an ideal modality to fight cancer, and the nature of photosensitizer limits its application in clinical therapy. The aim of this study was to explore a novel mode of drug delivery for the intervention of bile duct cancer.MethodsOxaliplatin and photosensitizer HCE6 were loaded with mesoporous silica nanoparticles (MSNs) to synthesize Oxaliplatin/HCE6-MSNs (OH-MSNs); the structure of OH-MSNs was characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS), the drug release rate was detected by high performance liquid chromatography; the cellular activity, apoptosis level, and the expression levels of intracellular apoptosis and autophagy-related factors of OH-MSNs on cholangiocarcinoma cells were observed by CCK-8, flow cytometry, colony formation assay, and Western blot; the effects of OH-MSNs on cholangioma growth were observed by mouse tumor formation, immunohistochemistry, and tissue Tunel staining.ResultsThe release of OH-MSNs to Oxaliplatin was enhanced under acidic conditions; compared with Oxaliplatin or O-MSNs, OH-MSNs showed more potent killing effects against cholangiocarcinoma cells (P<0.05), and exerted notably inhibitory effects on the activity of cholangiocarcinoma cells (P<0.05), promoted their apoptosis (P<0.05), and greatly facilitated the expression of pro-apoptotic factors and autophagic factors in cholangiocarcinoma cells (P<0.05), and markedly inhibited the expression of anti-apoptotic factors and autophagic inhibitory factors (P<0.05); moreover, OH-MSNs could significantly suppress the growth of mouse cholangiocarcinoma (P<0.05) and induce apoptosis of tumor cells compared with Oxaliplatin or O-MSNs (P<0.05).ConclusionMSNs loading greatly increases the killing effect of Oxaliplatin on cholangiocarcinoma cells and upgrades the autophagic level of cholangiocarcinoma cells, while OH-MSNs synthesized by further loading HCE6 have a more apparent killing effect on cholangiocarcinoma cells.

The role of NLRP3 in lead-induced neuroinflammation and possible underlying mechanism

BackgroundNeuroinflammation induced by lead exposure (Pb) is a major cause of neurotoxicity of Pb in the central nervous system (CNS). The NLR family, domain of pyrin containing 3 (NLRP3) involves in various neurological diseases, while the question of whether NLRP3 plays a role in lead-induced neuroinflammation has not yet been reported. MethodsDevelopmental and knockout (KO) NLRP3 mice were used to establish two in vivo models, and BV2 cells were used to establish an in vitro model. Behavioral and electrophysiologic tests were used to assess the neurotoxicity of Pb, and immunofluorescence staining was used to assess neuroinflammation. Real-time PCR and western blot were performed to examine the mRNA and protein levels of inflammatory cytokines and NLRP3 inflammasomes. siRNA technology was used to block NLRP3 expression. ResultsPb exposure led to neural injure and microglial activation in the hippocampus region, while minocycline intervention attenuated Pb-induced neurotoxicity by inhibiting neuroinflammation. Pb increased the expression of NLRP3 and promoted cleavage of caspase-1 in mRNA and protein levels, and minocycline partially reversed the effects of Pb on NLRP3 inflammasomes. Blocking of NLRP3 by KO mice or siRNA attenuated neural alterations induced by Pb, weakened microglial activation in vivo and in vitro as well, without affecting the accumulation of Pb. Pb increased autophagic protein levels and phosphorylation of NF-κB, while suppressing autophagy or NF-κB inhibited Pb's effects on NLRP3. ConclusionsNLRP3 is involved in the regulation of Pb-induced neurotoxicity. These findings expand mechanism research of Pb neurotoxicity and may help establish new prevention strategies for Pb neurotoxicity.

Promoting effect of rapamycin on osteogenic differentiation of maxillary sinus membrane stem cells.

BackgroundStem cells located in the maxillary sinus membrane can differentiate into osteocytes. Our study aimed to evaluate the effect of rapamycin (RAPA) on the osteogenic differentiation of maxillary sinus membrane stem cells (MSMSCs).MethodsColony-forming unit assay, immunophenotype identification assay, and multi-differentiation assay confirmed characteristics of MSMSCs obtained from SD rats. Transmission electron microscopy (TEM) and flow cytometry (FCM) identified the initial autophagic level of MSMSCs induced by RAPA. Real-time quantitative PCR (qPCR) evaluated subsequent autophagic levels and osteogenic differentiation. Alkaline phosphatase (ALP) activity assay and alizarin red staining (ARS) evaluated subsequent osteogenic differentiation. We performed a histological examination to clarify in vivo osteogenesis with ectopic bone mass from BALB/c nude mice.ResultsMSMSCs possessed an active proliferation and multi-differentiation capacity, showing a phenotype of mesenchymal stem cells. The autophagic level increased with increasing RAPA (0, 10, 100, 1,000 nM) and decreased over time. ALP activity and calcium nodules forming in four RAPA-treated groups on three-time points (7, 14, 21 d) showed significant differences. Col1a1, Runx2, and Spp1 expressed most in 100 nM RAPA group on 7 and 14 d. Osteogenesis-related genes except for Ibsp expression between four groups tended to be consistent on 21 d. 100 nM and 10 nM RAPA-treated groups showed more bone formation in vivo.ConclusionRAPA can promote osteogenic differentiation of MSMSCs, indicating a possible relationship between osteogenic differentiation and autophagy.

Selective S1PR 1 Ligand Attenuates Autophagy Mediated Human Cerebrovascular Smooth Muscle Cell Death Secondary To In Vitro Ischemic Injury

Cerebrovascular pathologies contribute significantly to acute ischemic stroke (AIS) outcome. Sphingosine-1-phosphate receptor (S1PR) modulators show beneficial effects against hypoxia plus glucose deprivation (HGD; in vitro ischemic-like model)-induced brain microvascular endothelial dysfunction and demonstrate clinical promise in improving cognitive AIS outcome in patients. However, the impact of S1PR modulation on vascular smooth muscle (VSM) health during ischemic injury has not been clearly elucidated. Therefore, the aim of this study was to investigate human brain VSM cell viability and autophagic responses following HGD. Adult male brain VSM cells were treated with ozanimod (selective S1PR type 1 ligand; 0.5nM) at P7 and exposed to normoxia (21% O2) or HGD (1% O2) for 9h, a timepoint based on cell survival studies conducted using trypan blue exclusion staining following HGD 3, 6, and 9h. Crystal violet staining determined vacuole presence and autophagic protein levels were examined using standard immunoblotting. Localization of autophagic markers were assessed via immunocytochemical labeling. We observed that HGD temporally decreased VSM cell viability and concomitantly increased vacuolization, and these responses were reversed by ozanimod. To characterize whether these vacuoles were of autophagosome phenotype, localization and punctation quantification of the upstream ubiquitin-like complex I mediator, Atg5 and its associated downstream autophagic marker, LC3B, were assessed. Following HGD insult, increased density of Atg5 and LC3B punctate clusters were visualized and expression levels were increased. In the presence of ozanimod, HGD-induced LC3B levels were attenuated suggesting that S1PR1modulation may regulate HGD-induced vacuole formation during autophagic flux. Concomitantly, basal levels of the anti-apoptotic and anti-autophagic protein Bcl-2 were significantly decreased following HGD, and ozanimod reversed this response. Further investigation revealed that while HGD did not alter LC3A/B-I (non-autophagic) levels, HGD-induced LC3A/B-II (autophagic) levels were increased but were attenuated by ozanimod. Autophagic protein levels of beclin-1 and Atg7 were not altered following HGD. However, in the presence of HGD plus ozanimod, we observed a decrease in Atg7 levels suggesting that ozanimod may have a protective effect against autophagy during HGD. While we did not observe alterations in beclin-1 protein levels across treatment groups, when combined with the decreased Bcl-2 levels, the availability of beclin-1 to mediate initiation of autophagy is enhanced following HGD via suppression of Bcl-2 inhibition. The rescue of Bcl-2 suggests that ozanimod may be suppressing the ability of beclin-1 to mediate initiation of autophagy following HGD. In conclusion, S1PRtype 1 ligands promote cell survival in part by attenuating HGD-induced autophagic flux in human brain VSM.

Discoid meniscus: High levels of apoptotic and autophagic genes.

How the meniscus adapts to the morphological changes in the lateral tibiofemoral compartment, in terms of gene expression, was the reason to establish this present study. This study aimed to determine the changes in the mRNA levels of the apoptotic and autophagic genes in the discoid meniscus. We have investigated the apoptotic and autophagic gene levels in discoid and normal lateral menisci of 21 patients (11 discoid and 10 control). The RNAs were isolated from the fresh discoid and healthy meniscal tissue. Gene expression was defined based on the threshold cycle (Ct), and Actin beta was used as a reference gene that acts as an internal reference to normalize RNA expression, which was calculated as 2-ΔΔCT. Apoptotic and autophagic gene levels were significantly higher in the discoid meniscus group. In discoid meniscus samples, the Bcl-2 mRNA, BclXL, BAK mRNA, ATG 12, ATG 7, ATG 5, ATG 3, and Beclin1 mRNA levels were higher by 4.2, 5.9, 9.1, 8.3, 23.2, 6.1, 12.4, and 18.1 times, respectively, with statistically significant differences (p < 0.001). The discoid meniscus etiology should be considered both in morphological and genetic modulation manners: apoptotic and autophagic genes play roles with tibiofemoral morphological differences.

Autophagy contributes to angiotensin II induced dysfunction of HUVECs

ABSTRACT Background: Signal transduction of Angiotensin II (Ang II) induced autophagy and its role in Ang II–induced dysfunction of HUVECs are still unclear. Methods: HUVECs are stimulated with different doses of Ang II (10-9-10-5 mol/L) for different time (6–48 hours). Autophagy-related protein markers: LC3, Beclin-1 and SQSTM1/p62 are measured by western blot. Results: Incubation with Ang II increases autophagic flux (Beclin-1, autophagosomes formation, and degradation of SQSTM1/p62, LC3-I). Increased autophagic levels are inhibited by pretreatment with Ang II type 1 receptor (AT1) blocker (Candesartan), NADPH Oxidase inhibitor (apocycin), mitochondrial KATP channels inhibitor (5-hydroxydecanoate, 5HD). 3-Methyladenine (inhibitors of autophagy) and rapamycin (activator of autophagy) respectively inhibits or activates Ang II–induced autophagy levels. Ang II decreases phosphorylation of endothelial nitric oxide synthase (eNOS) and NO production in HUVECs. L-NAME (NOS inhibitor) totally mimics the actions of Ang II on eNOS, NO production and autophagy levels. Rapamycin further decreases NO production combined with Ang II. Silence Atg5 completely reverses Ang II-activated autophagy levels. Conclusions: Our results demonstrate that Ang II stimulation increases autophagy levels via AT1 receptor, NADPH oxidase, mitochondrial KATP channel, eNOS, Atg5 signal pathway in HUVECs, and activation of autophagy contributes to Ang II induced dysfunction of HUVECs.

Activation of CREB-mediated autophagy by thioperamide ameliorates β-amyloid pathology and cognition in Alzheimer's disease.

Alzheimer's disease (AD) is an age‐related neurodegenerative disease, and the imbalance between production and clearance of β‐amyloid (Aβ) is involved in its pathogenesis. Autophagy is an intracellular degradation pathway whereby leads to removal of aggregated proteins, up‐regulation of which may be a plausible therapeutic strategy for the treatment of AD. Histamine H3 receptor (H3R) is a presynaptic autoreceptor regulating histamine release via negative feedback way. Our previous study showed that thioperamide, as an antagonist of H3R, enhances autophagy and protects against ischemic injury. However, the effect of thioperamide on autophagic function and Aβ pathology in AD remains unknown. In this study, we found that thioperamide promoted cognitive function, ameliorated neuronal loss, and Aβ pathology in APP/PS1 transgenic (Tg) mice. Interestingly, thioperamide up‐regulated autophagic level and lysosomal function both in APP/PS1 Tg mice and in primary neurons under Aβ‐induced injury. The neuroprotection by thioperamide against AD was reversed by 3‐MA, inhibitor of autophagy, and siRNA of Atg7, key autophagic‐related gene. Furthermore, inhibition of activity of CREB, H3R downstream signaling, by H89 reversed the effect of thioperamide on promoted cell viability, activated autophagic flux, and increased autophagic‐lysosomal proteins expression, including Atg7, TFEB, and LAMP1, suggesting a CREB‐dependent autophagic activation by thioperamide in AD. Taken together, these results suggested that H3R antagonist thioperamide improved cognitive impairment in APP/PS1 Tg mice via modulation of the CREB‐mediated autophagy and lysosomal pathway, which contributed to Aβ clearance. This study uncovered a novel mechanism involving autophagic regulating behind the therapeutic effect of thioperamide in AD.

RETRACTED ARTICLE: Two Metal–Organic Frameworks: Luminescent Detection Performance and Treatment Activity on Chronic Glomerulus Nephritis Mediated by E. coli Abundance Regulation

Two new metal–organic frameworks (MOFs) [Eu(Hpta)(C2O4)]·3H2O (1) and {[Co(pta)]·1.5H2O}n (2) were prepared via reaction of the corresponding metal salts with a bifunctional organic ligand 2-(4-pyridyl)-terephthalic acid (H2pta) under the solvothermal reaction conditions. The Eu(III)-based complex 1 is highly selective and sensitive to Cu2+ in the water solution, and nearly 93% of its fluorescent intensity could be quenched when the Cu2+ ion concentration is 10–2 mol/L. Complex 1 has a relative high quenching constant KSV of 1163 M−1,which might be highly related to its uncoordinated nitrogen sites for the binding with Cu2+ ion in aqueous solution as reflected by the X-ray photoelectron spectroscopy (XPS) analysis (from 399.6 eV to 400.2 eV). Furthermore, they were used for the chronic glomerulus nephritis treatment, the biological activity and detail mechanism was investigated. The CFU assay was implemented to determine the fecal bacterial numbers of Escherichia coli (E. coli) after treated with compound 1 or 2. Afterward, the western blot was implemented to determine the autophagic levels in the renal tubular endothelial cells.

Resveratrol attenuates cigarette smoke induced endothelial apoptosis by activating Notch1 signaling mediated autophagy

BackgroundIncreasing evidence shows that endothelial apoptosis contributes to cigarette smoke (CS)-induced disease progression, such as chronic obstructive pulmonary disease (COPD). Our previous studies have validated Notch1 as an anti-apoptotic signaling in CS-induced endothelial apoptosis. Resveratrol (RESV) is a naturally occurring polyphenol that exhibits an anti-apoptotic activity in endothelial cells that exposed to many kinds of destructive stimulus. However, the effects of resveratrol on Notch1 signaling in CS-induced endothelial apoptosis have not yet been fully elucidated. Therefore, the aim of this study was to examine whether RESV can protect endothelial cells from CS-induced apoptosis via regulating Notch1 signaling.MethodsHuman umbilical vein endothelial cells (HUVECs) were pretreated with RESV for 2 h, followed by cotreatment with 2.5%CSE for 24 h to explore the role of RESV in CSE induced endothelial apoptosis. 3-methyladenine (3-MA) or rapamycin was used to alter autophagic levels. Lentivirus Notch1 intracellular domain (LV-N1ICD), γ-secretase inhibitor (DAPT) and Notch1 siRNA were used to change Notch1 expression. The expression of Notch1, autophagic and apoptotic markers were examined by Western blot and the apoptosis rate was detected by Flow cytometry analysis.ResultsOur results showed that activating autophagy reduced CSE-induced endothelial apoptosis, while blocking autophagy promoted cell apoptosis in HUVECs. RESV pretreatment attenuated the CSE-induced endothelial apoptosis and activated Notch1 signaling. RESV pretreatment also increased LC3b-II and Beclin1 production, decreased p62 and mTOR expression. 3-MA treatment inhibited autophagy and aggravated CSE induced apoptosis, while rapamycin promoted autophagy, led to a decrease in cell apoptosis. LV-N1ICD transfection upregulated autophagy and reduced apoptosis. However, this protective effect was abolished by 3-MA treatment. In cells treated with DAPT or Notch1 siRNA, autophagy was decreased, while apoptosis was increased. RESV partly rescued the DAPT or Notch1 siRNA induced apoptosis by activating Notch1 signaling.ConclusionIn HUVECs, RESV attenuates CSE induced endothelial apoptosis by inducing autophagy in a Notch1-dependent manner.

The apoptotic and autophagic effects of cast Au-Pt, and differently manufactured Co-Cr and cp-Ti on three-dimensional oral mucosal model

The application of digitally manufactured dental metals has aroused the attention on their biocompatibilities. Three-dimensional oral mucosal model (3D OMM) would provide excellent assessments to the biocompatibility. In the current study, we set to measure metal ion release levels in the extracts of cast gold-platinum alloy (Au-Pt), differently manufactured cobalt-chromium alloy (Co-Cr) and commercially pure titanium (cp-Ti). We further tested two scaffold materials of 3D OMM to determine the better one for the succedent work. Lastly, we evaluated the apoptotic and autophagic effects of cast Au-Pt, and differently manufactured Co-Cr and cp-Ti on mucosal cells based on 3D OMM. We found that, in the construction of 3D OMM, Matrigel showed better performance than bovine acellular dermal matrix. Thus, Matrigel was chosen to construct the 3D OMM in the succedent studies. The results of ion release and biological assessments showed that, firstly, cast Au-Pt and cp-Ti triggered less early apoptotic cells and ion release than cast Co-Cr, implying better chemical stability and biocompatibility of them; secondly, digitally manufactured (including CAD/CAM milling and SLM) Co-Cr showed significantly lower ion release levels and lesser early apoptotic effects on 3D OMM as compared to the cast one. Although cast cp-Ti released much more ions than CAD/CAM milling one, manufacturing methods had no impact on apoptotic effect of cp-Ti. Therefore, we believe that digital methods possess same or even better chemical stability and biocompatibility than conventional casting one. Thirdly, although increased autophagic levels are observed in all test groups, so far there is no evidence that the test metals trigger different levels of autophagy as compared to each other. In addition, correlation analysis indicates that Co, W, and Mn appear to be the potential inducements for the apoptotic and autophagic effects of Co-Cr.

Fisetin Inhibits Autophagy in HepG2 Cells via PI3K/Akt/mTOR and AMPK Pathway

The effect of fisetin on autophagy in hepatocellular carcinoma remains uncovered. HepG2 cells were exposed to different concentrations of fisetin (25, 50, and 100 µM) for 24 h. The cells were also treated with rapamycin and chloroquine alone or in combination with fisetin. Autophagic flux formation and ATP levels were determined. The changes in autophagic markers and AMPK signaling proteins were analyzed using qRT-PCR and Western blotting. Cyto-ID staining followed by flow cytometry showed that fisetin decreased autophagic flux formation in a dose-dependent manner. In gene expression analysis, the mRNA levels of mTOR, Atg5, Atg16L, and LC3A were elevated, whereas the mRNA levels of Atg7 and Beclin1 were downregulated in a dose-dependent manner compared to control. In the Western blotting analysis, fisetin treatment inhibited the expression of Atg7, Atg16L, mTOR, and pACC and elevated the expression of Atg5, AMPKα, AMPKβ1/2, ACC and Akt. Taken together, the results revealed that fisetin inhibited autophagy by the activation of PI3K/Akt/mTOR and modulation of AMPK signaling pathways. Our findings indicate that suppression of autophagy by fisetin may serve as an effective therapeutic strategy against HCC.

Abstract 596: Endolysosomal trafficking defects leads to caspase independed cell death in colon cancer

Abstract Currently approved conventional chemotherapeutics for treating colorectal cancer (CRC) exert their effect through activating apoptotic pathways in cancer cells. However, CRC cells often become resistant due to mutations in apoptotic pathways rendering chemotherapeutics ineffective. Drug resistant CRC typically advances to metastatic stage, resulting in poor prognosis and shorter survival time. We have recently discovered structurally constrained 4-pyridynyl BAPT compounds that cause a form of non-apoptotic cell death in CRC cells characterized by the simultaneous induction of dysregulated macropinocytosis (i.e. self-drinking) and macroautophagy (i.e. self-eating). We define this distinctive type of caspase-independent cell death as ‘methuophagy' (from the Greek ‘methuo', to drink to intoxication, and ‘phagy', self-eating). In particular, our initial lead, BAPT-27, does not induce cell death via activation of the classical apoptotic pathways, but causes methuophagy in submicromolar range. BAPT-27 induces substantial accumulation of fluid-filled, heterogeneous vacuoles and autophagosomes, resulting in metabolic failure, loss of membrane integrity, and detachment of cells from the substratum. The cell death is accompanied by extensive autophagy in the absence of cell shrinkage, chromatin condensation, alterations in mitochondrial membrane potential, or damage to the nuclear membrane. Other hit analogues i.e. BAPT-36, BAPT-38, BAPT-54 also potently trigger this form of non-apoptotic cell death in CRC cells. We observed that methuophagic cell death is accompanied by a significant increase in lysosomal activity, numbers, and biogenesis as confirmed by ultrastructural imaging. Our results suggest that BAPT analogues produce endolysosomal trafficking defects that prevent recycling of lysosomes and cause lysosome-to-nucleus signaling defect, a mechanism that disturb cellular clearance and energy metabolism. Furthermore, they disrupt vesicular trafficking at the lysosomal nexus, manifested by defective processing of procathepsins, impairment of autophagic flux, V-ATPase levels, and accumulation of autophagosomes and acrgo protein p-62. However, specific changes in core pharmacophore could result in loss of methuophagy induction and drive the cell death towards apoptosis. For example, BAPT-42 was found to have limited effects on lysosomal pathways and does not kill the cells through methuophagy, but induces apoptosis. Identifying the biology of methuophagy-induced cell death and lysosomal clearance pathways will allow us to better design this new class of compounds that kill CRC cells via non-apoptotic mechanisms for treatment of patients with apoptotic-resistant and aggressive CRCs. Citation Format: Noor Hussein, Mariah Pasternak, Shikha Kumari, Zaynah Awthe, William Gunning, Paul C. Trippier, Amit Tiwari. Endolysosomal trafficking defects leads to caspase independed cell death in colon cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 596.