Light Chain 3A Research Articles

Immunoexpression of Autophagy-Related Proteins in Salivary Gland Tumors: An Exploratory Study.

Autophagy is a cellular survival mechanism involved in several human diseases, but its participation in the development of salivary gland tumors is not fully understood. This study investigated the immunoexpression of autophagy-related proteins (autophagy-related 7 [Atg7], microtubule-associated protein 1 light chain 3A [LC3A], microtubule-associated protein 1 light chain 3B [LC3B], protein p62 [p62], and phosphorylated mammalian target of rapamycin [p-mTOR]) in pleomorphic adenoma (PA), polymorphous adenocarcinoma (PAC), mucoepidermoid carcinoma (MEC), and adenoid cystic carcinoma (ACC) of salivary glands. Twenty PAs, 20 PACs, 20 MECs, and 14 ACCs were selected. The percentages of cytoplasmic and nuclear positivity for autophagy-related proteins in neoplastic cells were assessed and correlated with histopathological parameters. Cytoplasmic immunoexpression of Atg7 was observed in all groups, with high median percentages of positivity. Regarding LC3A and LC3B, cytoplasmic immunoexpression was found in most PACs (95%) and in all cases of PA, MEC and ACC, with the highest percentages of positivity in PACs and PAs (p < 0.005). ACCs exhibited lower cytoplasmic immunoexpression of p-mTOR (p < 0.005) and lower nuclear expression of p62 (p < 0.05) when compared to PAs, PACs and MECs. Low nuclear immunoexpression of Atg7, LC3A and p-mTOR and absence of nuclear staining for LC3B were observed in all groups. Regarding histopathological parameters of PAs, MECs and ACCs, there were no significant differences in the expression of autophagy-related proteins. In all groups, positive correlations were observed between the immunoexpression of some autophagy-related proteins (p < 0.05). The results suggest the participation of autophagy in the pathogenesis of PA, PAC, MEC, and ACC of salivary glands. Upregulation of autophagy and reduced nuclear translocation of p62 may contribute to the aggressive biological behavior of salivary gland ACC.

Total Chemical Synthesis of LC3A and LC3B Activity-Based Probes

Autophagy is a conserved cellular process involved in the degradation of intercellular materials. During this process, double-membrane vesicles called autophagosomes engulf cytoplasmic components ready for degradation. A key component in the formation of autophagosomes are the autophagy-related (Atg) proteins, including microtubule-associated protein light chain 3A (LC3A) and 3B (LC3B). After the C-terminus of LC3 is conjugated to a phospholipid, it promotes the elongation of the phagosome and provides a docking station for the delivery of proteins ready for degradation. Since dysregulation of the autophagy pathway has been associated with a variety of human diseases, components of this process have been considered as potential therapeutic targets. However, the mechanistic details of LC3-specific ligases and deconjugation enzymes are far from unraveled and chemical tools for activity profiling could aid in affording more insights into this process. Herein, we describe a native chemical ligation approach for the synthesis of two LC3 activity-based probes (ABPs). Initial studies show that the probes covalently interact with the cysteine protease ATG4B, showcasing the potential of these probes to unravel mechanistic and structural details.

Vitamin D receptor deficiency increases systolic blood pressure by upregulating the renin-angiotensin system and autophagy.

The vitamin D receptor (VDR) may regulate blood pressure via multiple pathways. The present study investigated the underlying mechanism by which VDR deficiency increases blood pressure. A total of 16 8-week-old male littermate mice were randomly divided into the VDR knockout and wild-type groups (VDR-/- and VDR+/+, respectively). Blood pressure was measured using a four-channel PowerLab data acquisition and ADI software analysis system. After euthanasia, vascular smooth muscle cells (VSMCs) were isolated from the VDR-/- and VDR+/+ mice. Oxidative stress, renin-angiotensin system (RAS) activation and autophagy markers were measured in the isolated VSMCs using reverse transcription-quantitative PCR (RT-qPCR), western blotting and transmission electron microscopy (TEM) assays. Mean systolic pressure was significantly higher in the VDR-/- mice compared with the VDR+/+ mice. RT-qPCR and western blotting analyses indicated that RAS markers (angiotensin II and II type 1 receptor) were significantly upregulated, oxidative stress was increased (evidenced by reduced superoxide dismutase and peroxiredoxin-4) and autophagy was activated (upregulation of autophagy related protein 7, Beclin 1 and microtubule-associated proteins 1A/1B light chain 3A) in the VDR-/- VSMCs compared with the VDR+/+ VSMCs. TEM demonstrated that there were more autophagy bodies in the VDR-/- VSMCs compared with the VDR+/+ VSMCs. In conclusion, VDR deficiency was associated with high blood pressure. The mechanism underlying the increase in blood pressure caused by VDR deficiency may involve activation of the RAS, as well as increased oxidative stress and autophagy of VSMCs.

Evaluation of the protective effects of morin against acrylamide-induced lung toxicity by biomarkers of oxidative stress, inflammation, apoptosis, and autophagy.

Acrylamide (ACR) has genotoxic, neurotoxic, and carcinogenic effects. From past to present, various plants or their products have been used for therapeutic purposes such as morin. It was aimed to detect possible protective effects of morin vs ACR-induced lung toxicity. The rats, treated with ACR alone or with morin for 10 consecutive days, were included in the study. A broad variety of biomarkers related to oxidative stress, apoptosis, autophagy, and inflammatory responses were evaluated. ACR increased malondialdehyde (MDA), tumor necrosis factor-alpha (TNF-α), cyclooxygenase-2 (COX-2), nuclear factor kappa-B (NF-κB), Beclin-1, IL-1β, bcl-2 associated X protein (Bax), caspase-3, light chain 3-A (LC3-A), and light chain 3-B (LC3-B) levels but reduced mammalian target of rapamycin (mTOR), b-cell lymphoma-2 (Bcl-2), catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), glutathione (GSH) in lung tissues. The morin had effects on the level of these molecules in a way that is opposite to ACR. While ACR-induced oxidative stress, apoptotic, autophagic, inflammatory responses, and may cause pulmonary dysfunction, the morin reduced ACR-induced lung damage. PRACTICAL APPLICATIONS: ACR is a toxic chemical produced by frying, baking, roasting, or grilling foods with high starch content and has genotoxic, neurotoxic, and carcinogenic effects. As an antioxidant compound, the morin is obtained from plants or their products. It was aimed to detect possible protective effects of morin against ACR-induced lung toxicity. It was detected that ACR-induced oxidative stress, apoptotic, autophagic, inflammatory responses, and may cause pulmonary dysfunction, but the morin reduced ACR-induced lung damage.

ASPM promotes hepatocellular carcinoma progression by activating Wnt/β-catenin signaling through antagonizing autophagy-mediated Dvl2 degradation.

Hepatocellular carcinoma (HCC) is one of the most fatal cancers worldwide. In this article, we show that expression of abnormal spindle‐like microcephaly‐associated protein (ASPM) is up‐regulated in liver cancer samples, and this up‐regulation is significantly associated with tumor aggressiveness and reduced survival times of patients. Down‐regulation of ASPM expression inhibits the proliferation, invasion, migration and epithelial‐to‐mesenchymal transition of HCC cells in vitro and inhibits tumor formation in nude mice. ASPM interacts with disheveled‐2 (Dvl2) and antagonizes autophagy‐mediated Dvl2 degradation by weakening the functional interaction between Dvl2 and the lipidated form of microtubule‐associated proteins 1A/1B light chain 3A (LC3II), thereby increasing Dvl2 protein abundance and leading to Wnt/β‐catenin signaling activation in HCC cells. Thus, our results define ASPM as a novel oncoprotein in HCC and indicate that disruption of the Wnt–ASPM–Dvl2–β‐catenin signaling axis might have potential clinical value.

Diosgenin ameliorates cellular and molecular changes in multiple sclerosis in C57BL/6 mice

Multiple sclerosis (MS) is especially known as a demyelinating disease of the central nervous system. Current treatments for MS are mostly based on controlling neuroinflammation and there are no treatments to promote the remyelination process at present. Diosgenin is a known herbal anti-inflammatory and antioxidant agent, which has also been shown to stimulate the growth of myelin in vitro. However, there is no or little evidence about diosgenin effects; specially on myelination, neuroprotection and its corresponding mechanisms in vivo in experimental autoimmune encephalomyelitis (EAE) as the most valid experimental model of MS. In this study, the therapeutic effect of diosgenin on clinical signs of EAE, and the corresponding cellular and molecular mechanisms have been examined with emphasis on myelination and neuroprotection mechanisms. EAE was induced using myelin oligodendrocyte glycoprotein (MOG) antigen in C57BL/6 mice. Diosgenin was gavaged (100 mg/kg) daily with the onset of paralysis signs (half tail paralysis) until the 18th post-immunization day in the treatment group. Blood and spinal cord tissue sampling was performed on post-immunization day 18. Lumbar spinal cord inflammation, demyelination, and axonal degeneration were assessed using Hematoxylin and Eosin (H & E), Luxol Fast Blue (LFB), and Bielschowsky's silver staining methods, respectively. Serum and spinal cord tissue level of tumor necrosis factor alpha (TNFα) and tissue levels of matrix metalloproteinase 9 (MMP-9) and interleukin 17 (IL-17) as inflammatory markers, microtubule-associated proteins 1A/1B light chain 3A (MAP1LC3A), and activity dependent neuroprotector homeobox (ADNP) as neuroprotective markers were assayed using enzyme linked immunosorbent assay (ELISA) method. The clinical score of EAE in the diosgenin treatment group was significantly reduced compared to the EAE group on days 15 to 18 after induction of the EAE (p < 0.001). Inflammation, demyelination and axonal loss scores also decreased significantly in the diosgenin treatment group compared to the EAE group (p < 0.05). Serum and spinal cord tissue level of TNFα and tissue level of MMP-9 considerably decreased in the diosgenin treatment group in comparison with the EAE group (p < 0.01). Diosgenin treatment had no significant effects on the tissue levels of IL-17, ADNP and MAP1LC3A. Therefore, diosgenin improved the clinical signs of EAE through lowering neuroinflammation, demyelination and axonal degeneration, but did not significantly affect the neuroprotective factors in this study. As a result, diosgenin could be a good candidate for new MS treatment strategies that, in addition to their anti-inflammatory effects, also enhance myelination.

Potential protective effect of hesperidin on hypoxia/reoxygenation-induced hepatocyte injury.

Hesperidin (HDN) has been reported to have hydrogen radical- and hydrogen peroxide-removal activities and to serve an antioxidant role in biological systems. However, whether HDN protects hepatocytes (HCs) against hypoxia/reoxygenation (H/R)-induced injury remains unknown. The present study aimed to explore the role of HDN in H/R-induced injury. HCs were isolated and cultured under H/R conditions with or without HDN treatment. HC damage was markedly induced under H/R, as indicated by cell viability, supernatant lactate dehydrogenase levels and alanine aminotransferase levels; however, HDN treatment significantly reversed HC injury. Oxidative stress markers (malondialdehyde, superoxide dismutase, glutathioneand reactive oxygen species) were increased markedly during H/R in HCs; however, this effect was significantly attenuated after exposure to HDN. Compared with those of the control group, the mRNA expression levels of IL-6 and TNF-α in HCs and the concentrations of IL-6 and TNF-α in the supernatants increased significantly following H/R, and HDN significantly ameliorated these effects. Western blotting demonstrated that microtubule-associated protein 1 light chain 3α (MAP1LC3A, also known as LC3) and Beclin-1 protein expression levels increased, while sequestosome 1 levels decreased during H/R following exposure to HDN. The number of GFP-LC3 puncta in HCs following exposure to HDN was increased compared with that observed in HCs without HDN exposure under the H/R conditions after bafilomycin A1 treatment. In summary, the present study demonstrated that HDN attenuated HC oxidative stress and inflammatory responses while enhancing autophagy during H/R. HDN may have a potential protective effect on HCs during H/R-induced injury.

Morin protects against acrylamide-induced neurotoxicity in rats: an investigation into different signal pathways.

The presented study investigates the effects of morin against toxicity induced by acrylamide (ACR) in the brains of Sprague Dawley rats. In this study, neurotoxicity was induced by orally administering 38.27 mg/kg/b.w ACR to rats through gastric gavage for 10 days. Morin was administered at the same time and at different doses (50 and 100 mg/kg/b.w) with ACR. Biochemical and Western blot analyses showed that ACR increased malondialdehyde (MDA), p38α mitogen-activated protein kinase (p38α MAPK), nuclear factor kappa-B (NF-κB), tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), cyclooxygenase-2 (COX-2), p53, caspase-3, bcl-2 associated X protein (Bax), Beclin-1, light chain 3A (LC3A), and light chain 3B (LC3B) levels and decreased those of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione (GSH), b-cell lymphoma-2 (Bcl-2), mammalian target of rapamycin (mTOR), phosphoinositide 3-kinase (PI3K), and protein kinase B (Akt) in brain tissue and therefore induced neurotoxicity by causing oxidative stress, inflammation, apoptosis, and autophagy. On the other hand, it was determined that morin positively affected the levels of these markers by displaying antioxidant, anti-inflammatory, anti-apoptotic, and anti-autophagic properties and had a protective effect on ACR-induced neurotoxicity. As a result, morin is an effective substance against brain damage caused by ACR, yet further studies are needed to use it effectively.

MiR‑143‑3p inhibits endometriotic stromal cell proliferation and invasion by inactivating autophagy in endometriosis.

Endometriosis (EM) is a multifactorial and debilitating chronic benign gynecological disease, but the pathogenesis of the disease is not completely understood. Dysregulated expression of microRNAs (miRNA/miR) is associated with the etiology of EM due to their role in regulating endometrial stromal cell proliferation and invasion. The present study aimed to identify the functions and mechanisms underlying miR-143-3p in EM. To explore the role of miR-143-3p in EM, functional miRNAs were analyzed via bioinformatics analysis. miR-143-3p expression levels in endometriotic stromal cells (ESCs) and normal endometrial stromal cells (NESCs) were measured via reverse transcription-quantitative PCR. The role of miR-143-3p in regulating ESC proliferation and invasion was assessed by performing Cell Counting Kit-8 and Transwell assays, respectively. miR-143-3p expression was significantly upregulated in ESCs compared with NESCs. Functionally, miR-143-3p overexpression inhibited ESC proliferation and invasion, whereas miR-143-3p knockdown promoted ESC proliferation and invasion. Moreover, miR-143-3p inhibited autophagy activation in ESCs, as indicated by decreased green puncta, which represented autophagic vacuoles, decreased microtubule associated protein 1 light chain 3α expression and increased p62 expression in the miR-143-4p mimic group compared with the control group. Moreover, compared with the control group, miR-143-3p overexpression significantly decreased the expression levels of autophagy-related 2B (ATG2B), a newly identified target gene of miR-143-3p, in ESCs. ATG2B overexpression reversed miR-143-3p overexpression-mediated inhibition of ESC proliferation and invasion. Collectively, the results of the present study suggested that miR-143-3p inhibited EM progression, thus providing a novel target for the development of therapeutic agents against EM.

Ginsenoside Rg2 protects cardiomyocytes against trastuzumab-induced toxicity by inducing autophagy.

Trastuzumab (TZM) significantly improves the outcomes of patients with breast cancer; however, it is associated with severe cardiotoxicity. Ginsenoside Rg2 was reported to exert protective effects against myocardial injury and apoptosis in human cardiomyocytes (HCMs). However, whether ginsenoside Rg2 protects HCMs against TZM-induced toxicity remains unclear. The present study investigated the proliferation of HCMs using a Cell Counting Kit-8 assay and Ki67 immunofluorescence staining. Apoptotic cells were detected by Annexin V/propidium iodide staining and flow cytometry. Furthermore, monodansylcadaverine staining was performed to detect cell autophagy. In addition, western blotting was used to detect the expression levels of phosphorylated (p)-Akt, p-mTOR, beclin 1, microtubule associated protein 1 light chain 3α (LC3) and autophagy protein 5 (ATG5) in HCMs. Pretreatment with ginsenoside Rg2 significantly protected HCMs against TZM-induced cytotoxicity by inhibiting apoptosis. Furthermore, pretreatment with ginsenoside Rg2 induced autophagy in HCMs by upregulating the expression levels of p-Akt, p-mTOR, beclin 1, LC3 and ATG5. The results obtained in the present study suggested that ginsenoside Rg2 could protect HCMs against TZM-induced cardiotoxicity by activating autophagy. Therefore, ginsenoside Rg2 may serve as a potential therapeutic agent to prevent TZM-related cardiotoxicity in patients with breast cancer.

Overexpression of lncRNA Dancr inhibits apoptosis and enhances autophagy to protect cardiomyocytes from endoplasmic reticulum stress injury via sponging microRNA-6324.

Endoplasmic reticulum stress (ERS) contributes to the pathogenesis of myocardial ischemia/reperfusion injury and myocardial infarction (MI). Long non-coding RNAs (lncRNAs) serve an important role in cardiovascular diseases, and lncRNA discrimination antagonizing non-protein coding RNA (Dancr) alleviates cardiomyocyte damage. microRNA (miR)-6324 was upregulated in MI model rats and was predicted to bind to Dancr. The present study aimed to investigate the role of Dancr in ERS-induced cardiomyocytes and the potential underlying mechanisms. Tunicamycin (Tm) was used to induce ERS. Cell viability, apoptosis and levels of associated proteins, ERS and autophagy in Dancr-overexpression H9C2 cells and miR-6234 mimic-transfected H9C2 cells were assessed using Cell Counting Kit-8, TUNEL staining and western blot assay, respectively. The results suggested that Dancr expression levels and cell viability were downregulated by Tm in a concentration-dependent manner compared with the control group. Tm induced apoptosis, ERS and autophagy, as indicated by an increased ratio of apoptotic cells, increased expression levels of Bax, cleaved (c)-caspase-3/9, glucose-regulated protein 78 kDa (GRP78), phosphorylated (p)-inositol-requiring enzyme-1α (IRE1α), spliced X-box-binding protein 1 (Xbp1s), IRE1α, activating transcription factor (ATF)6, ATF4, Beclin 1 and microtubule associated protein 1 light chain 3α (LC3)II/I, and decreased expression levels of Bcl-2, unspliced Xbp1 (Xbp1u) and p62 in the Tm group compared with the control group. Moreover, the results indicated that compared with the Tm + overexpression (Oe)-negative control (NC) group, the Tm + Oe-Dancr group displayed decreased apoptosis, but enhanced ERS and autophagy to restore cellular homeostasis. Compared with the Tm + Oe-NC group, the Tm + Oe-Dancr group decreased the ratio of apoptotic cells, decreased expression levels of Bax, c-caspase-3/9 and Xbp1u, and increased expression levels of Bcl-2, p-IRE1α, Xbp1s, Beclin 1 and LC3II/I. Dancr overexpression also significantly downregulated miR-6324 expression compared with Oe-NC. The Dual-luciferase reporter assay further indicated an interaction between Dancr and miR-6324. In addition, miR-6324 mimic partially reversed the effects of Dancr overexpression on Tm-induced apoptosis, ERS and autophagy. In conclusion, lncRNA Dancr overexpression protected cardiomyocytes against ERS injury via sponging miR-6324, thus inhibiting apoptosis, enhancing autophagy and restoring ER homeostasis.

Abstract 1830: Evaluation of anti-tumor effects of VER-155008, a functional inhibitor of HSP70, through macroautophagy in mesothelioma cells

Abstract Pleural mesothelioma is a poor prognostic malignancy because the effect of therapies remains limited; and thus, it is urgent to elucidate the pathophysiology leading to newer treatment. Our previous investigation showed that growth of mesothelioma cells was inhibited by suppression of dishevelled-3 on Wnt signaling pathway, accompanied with reduction of heat shock protein 70 (HSP70), which works as a cell-protective molecular chaperon against heat stress, oxidative stress, heavy metals and others, by refolding proteins. Furthermore, HSP70 has been showed to affect cell signaling such as Akt/mTOR pathway, which negatively regulates macroautophagy. This study evaluated effects of VER-155008, an adenosine-derived functional inhibitor of HSP70, on cell growth and macroautophagy of mesothelioma cell lines. Mesothelioma cell lines including MSTO-211H (211H), NCI-H2452 (H2452) and NCI-H28 (H28) were cultured with 0.5-40 µM VER-155008 for cell viability analysis by water-soluble tetrazolium salt-8, colony formation assay, cell cycle analysis and the following analyses. Western blotting confirmed the expression of HSP70, Akt, phosphorylated (p-) Akt, JNK, p-JNK, light chain 3A (LC3A) and lysosome-associated membrane protein 2A (LAMP2A). For evaluation of macroautophagy, a plasmid, pMRX-IP-GFP-LC3-RFP-LC3ΔG, was transfected to mesothelioma cells. The transfected cells generate GFP-LC3-RFP-LC3ΔG, and it was divided into GFP-LC3 and RFP-LC3ΔG by inherent ATG4. GFP-LC3 decreases by macroautophagy activation, while RFP-LC3ΔG serves as an internal control. VER-155008 suppressed cell viability dose-dependently, and colony formation of 211H, H2452 and H28. In 211H and H28 cells, the proportions of cell count in G1 phase significantly increased, accompanied with inhibition of cell growth. In western blot analysis, VER-155008 reduced expression of p-Akt, while the expression of Akt, p-JNK, JNK, LC3A, LAMP2A and HSP70 were not altered. In macroautophagy analysis by the use of mesothelioma cells, which were transfected with the plasmid and stably expressed GFP-LC3-RFP-LC3ΔG, 20 µM VER-155008, 5.0 µM gefitinib and deprivation of fetal bovine serum (FBS) induced macroautophagy. In addition, VER-155008 enhanced FBS-deprivation-induced macroautophagy. From these results, functional inhibition of HSP70 by VER-155008 suppressed cell growth in pleural mesothelioma cells, accompanied with enhancement of macroautophagy. Citation Format: Kosuke Sakai, Maya Inoue, Hiroaki Nishimura, Shintaro Mikami, Yoshiki Kuwabara, Akitoshi Kojima, Maiko Toda, Yumiko Ogawa, Satoshi Kikuchi, Yusuke Hirata, Yuriko Mikami, Hiroyuki Kyoyama, Gaku Moriyama, Akihiko Gemma, Kazutsugu Uematsu. Evaluation of anti-tumor effects of VER-155008, a functional inhibitor of HSP70, through macroautophagy in mesothelioma cells [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 1830.

Endoplasmic reticulum stress, unfolded protein response and autophagy contribute to resistance to glucocorticoid treatment in human acute lymphoblastic leukaemia cells.

Acute lymphoblastic leukaemia(ALL) is the most frequent childhood cancer and, although it is highly treatable, resistance to therapy, toxicity and side effects remain challenging. The synthetic glucocorticoid(GC) dexamethasone(Dex) is commonly used to treat ALL, the main drawback of which is the development of resistance to this treatment. The aim of the present study was to investigate potential molecular circuits mediating resistance and sensitivity to GC‑induced apoptosis in ALL. The leukaemia cell lines CEM‑C7‑14, CEM‑C1‑15 and MOLT4 treated with chloroquine(CLQ), thapsigargin(TG) and rotenone(ROT) were used to explore the roles of autophagy, endoplasmic reticulum(ER) stress/unfolded protein response(UPR) and reactive oxygen species(ROS) generation in the response to GC treatment. ROS levels were associated with increased cell death and mitochondrial membrane potential in rotenone‑treated CEM cells. Autophagy inhibition by CLQ exhibited the strongest cytotoxic effect in GC‑resistant leukaemia. Autophagy may act as a pro‑survival mechanism in GC‑resistant leukaemia since increasing trends in beclin‑1 and microtubule‑associated protein1 light chain3α levels were detected in CEM‑C1‑15 and MOLT4 cells treated with Dex, whereas decreasing trends in these autophagy markers were observed in CEM‑C7‑14 cells. The intracellular protein levels of the ER stress markers glucose‑regulated protein(GRP)78 and GRP94 were stimulated by Dex only in the GC‑sensitive cells, suggesting a role of these chaperones in the GC‑mediated ALL cell death. Increased cell surface levels of GRP94 were recorded in CEM‑C7‑14 cells treated with combination of Dex with TG compared with those in cells treated with TG alone, whereas decreasing trends were observed in CEM‑C1‑15 cells under these conditions. Taken together, the results of the present study demonstrated that autophagy may be a pro‑survival mechanism in GC‑resistant leukaemia, and by modulating intracellular and surface GRP94 protein levels, Dex is involved in the regulation of ER stress/UPR‑dependent cell death and immune surveillance. These observations may be of clinical importance if confirmed in patients.

The Carbonic Anhydrase Inhibitor Dorzolamide Stimulates the Differentiation of Human Meibomian Gland Epithelial Cells

ABSTRACT Purpose Clinical studies have indicated that the long-term use of topical antiglaucoma drugs, such as carbonic anhydrase inhibitors (CAIs), may lead to meibomian gland dysfunction (MGD). We hypothesize that these adverse effects involve a direct influence on human MG epithelial cells (HMGECs). The purpose our present investigation was to test our hypothesis and determine whether exposure to dorzolamide, a CAI, impacts the proliferation, intracellular signaling and differentiation of HMGECs. Materials and Methods We cultured immortalized (i) HMGECs with vehicle or various concentrations of dorzolamide for 6 days. Cells were enumerated with a hemocytometer, and examined for their morphology, Akt signaling activity, accumulation of neutral lipids, phospholipids and lysosomes, and the expression of protein biomarkers for lipogenesis regulation, lysosomes and autophagosomes. Results Our results show that a high, 500 µg/ml concentration of dorzolamide causes a significant decrease in Akt signaling and the proliferation of iHMGECs. However, the high dose of dorzolamide also promotes the differentiation of iHMGECs. This response features increases in the number of lysosomes, the accumulation of phospholipids, and the expression of the light chain 3A biomarker for autophagosomes. In contrast, the therapeutic amount (50 µg/ml) of dorzolamide has no impact on the proliferative or differentiative abilities of iHMGECs. Conclusions Our results support our hypothesis and demonstrate that the CAI dorzolamide does exert a direct influence on the proliferation and differentiation of iHMGECs. However, this effect is elicited only by a high, and not a therapeutic, amount of dorzolamide. Abbreviations: AKT: phosphoinositide 3-kinase-protein kinase B; BPE: bovine pituitary extract; CAD: cationic amphiphilic drug; DED: dry eye disease; DMEM/F12: 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F-12; EGF: epidermal growth factor; FBS: fetal bovine serum; iHMGECs: immortalized human meibomian gland epithelial cells; KSFM: keratinocyte serum-free medium; LAMP-1: lysosomal-associated membrane protein 1; LC3A: light chain 3A; MGD: meibomian gland dysfunction; SREBP-1: sterol regulatory element-binding protein 1

Excessive production of mitochondrion‑derived reactive oxygen species induced by titanium ions leads to autophagic cell death of osteoblasts via the SIRT3/SOD2 pathway

The incidence of peri-implant bone loss is high, and is a difficult condition to treat. Previous studies have shown that titanium (Ti) ions released from implants can lead to osteoblast cell damage, but the specific mechanisms have not been elucidated. The present study established a Ti ion damage osteoblast cell model. The levels of mitochondrion-derived reactive oxygen species (mROS) and autophagy, cell viability and the sirtuin 3 (SIRT3)/superoxide dismutase 2 (SOD2) pathway were examined in this model. It was found that Ti ions decreased osteoblast viability. Moreover, with increased Ti ion concentration, the expression levels of microtubule associated protein 1 light chain 3α (LC3) progressively increased, P62 decreased, autophagic flow increased and mROS levels increased. After the addition of an autophagy inhibitor Bafilomycin A1 and Mito-TEMPO, a mitochondrial antioxidant, the production of mROS was inhibited, the level of autophagy was decreased and cell activity was improved. In addition, with increased Ti ion concentration, the activity of SOD2 decreased, the acetylation level of SOD2 increased, the SIRT3 mRNA and protein expression levels decreased, and the activity of SIRT3 was significantly decreased. Furthermore, it was demonstrated that SIRT3 overexpression reduced the acetylation of SOD2 and increased the activity of SOD2, as well as reducing the production of mROS and the expression level of LC3, thus increasing cell viability. Therefore, the present results suggested that excessive production of mROS induced by Ti ions led to autophagic cell death of osteoblasts, which is dependent on the SIRT3/SOD2 pathway.

Anti-fibrosis activity of quercetin attenuates rabbit tracheal stenosis via the TGF-β/AKT/mTOR signaling pathway

AimsThis study aimed to explore the possible mechanism of trauma-induced laryngotracheal stenosis and potential protective and therapeutic efficacy of quercetin on trauma-induced laryngotracheal stenosis. Main methodsThe expression and activity of fibrotic factors [interleukin (IL)-6, IL-8, autophagy related 5 (ATG5), collagen (COL)-1, tumor growth factor (TGF)-β COL-3, microtubule-associated proteins 1A/1B light chain 3A (LC3), and vascular endothelial growth factor (VEGF)] and fibrotic signaling mediators [mammalian target of rapamycin (mTOR) and phosphorylated AKT (pAKT)] were detected by real-time quantitative PCR (qRT-PCR), ELISA, Western blot, and immunohistochemical staining, respectively, in the lipopolysaccharide (LPS)-induced WI-38 (a human embryonic lung fibroblast cell line) cellular fibrotic model and a trauma-induced rabbit tracheal stenosis model, with and without quercetin treatment. Key findingsPre-treatment with quercetin significantly reversed the LPS-induced upregulation of pro-fibrotic factors (IL-6, IL-8, COL-1, COL-3, LC3) and fibrotic signaling mediators (mTOR and AKT), and it induced the downregulation of ATG5 in the WI-38 cells. Furthermore, the anti-fibrotic activity of quercetin was confirmed in the trauma-induced rabbit tracheal stenosis model. Thus, the nasogastric administration of quercetin attenuated the tracheal stenosis of the rabbit tracheal stenosis model, in addition to effectively reversing an increase in pro-fibrotic factors (VEGF, IL-6, TGF-β, COL-1, and COL-3) and fibrotic signaling mediators (mTOR and AKT), as well as downregulating ATG5 of the rabbit tracheal stenosis model. SignificanceQuercetin exhibits anti-fibrotic activity by inhibiting pro-fibrotic factors and AKT/mTOR signaling pathway, in addition to activating autophagy activity. This study provided experimental evidence supporting the application of quercetin in tracheal stenosis, clinically.

β‑asarone modulates Beclin‑1, LC3 and p62 expression to attenuate Aβ40 and Aβ42 levels in APP/PS1 transgenic mice with Alzheimer's disease.

Alzheimer's disease (AD) is a common neurodegenerative disease in the elderly population. Autophagy is a well-known regulator of neurodegenerative diseases and β-asarone has been discovered to have certain neuropharmacological effects. Thus, the present study aimed to analyze the potential effects of β-asarone in AD and its possible mechanism of action in relation to autophagy. The present study investigated the effects of β-asarone on the number of senile plaques and amyloid β(Aβ)40, Aβ42, amyloid precursor protein (APP) and Beclin-1 mRNA levels in the hippocampus of APP/presenilin-1 (PS1) transgenic mice. The possible mechanism of β-asarone on autophagy-related proteins, including Beclin-1, light chain (LC)3A, LC3B and p62 levels, and the number of autophagosomes was also investigated. Mice were divided into a normal control group, a model group, a β-asarone-treated group, a 3-MA-treated group and a rapamycin-treated group. Treatments were continuously administered to all mice for 30 days by intragastric administration. The mice, including those in the normal and model control groups, were given equal volumes of saline. It was demonstrated that β-asarone treatment reduced the number of senile plaques and autophagosomes, and decreased Aβ40, Aβ42, APP and Beclin-1 expression in the hippocampus of model mice compared with untreated model mice. β-asarone also inhibited LC3A/B expression levels, but increased p62 expression. It was deduced that the neuroprotective effects of β-asarone in APP/PS1 transgenic mice resulted from its inhibition of autophagy. In conclusion, the data suggested that β-asarone should be explored further as a potential therapeutic agent in AD.

Effect of ATM on inflammatory response and autophagy in renal tubular epithelial cells in LPS-induced septic AKI.

The aim of the present study was to explore the role of ataxia-telangiectasia mutated (ATM) in lipopolysaccharide (LPS)-induced in vitro model of septic acute kidney injury (AKI) and the association between ATM, tubular epithelial inflammatory response and autophagy. The renal tubular epithelial cell HK-2 cell line was cultured and passaged, with HK-2 cell injury induced by LPS. The effects of LPS on HK-2 cell morphology, viability, ATM expression and inflammation were observed. Lentiviral vectors encoding ATM shRNA were constructed to knock down ATM expression in HK-2 cells. The efficiency of ATM knockdown in HK-2 cells was detected by western blot analysis and reverse transcription-quantitative PCR (RT-qPCR). HK-2 cells transfected with the ATM shRNA lentivirus were used for subsequent experiments. Following ATM knockdown, corresponding controls were set up, and the effects of ATM on inflammation and autophagy were detected in HK-2 cells using RT-qPCR, western blotting and ELISA. After LPS stimulation, the HK-2 cells were rounded into a slender or fusiform shape with poorly defined outlines. LPS treatment reduced cell viability in a partly dose-dependent manner. LPS increased the expression of tumor necrosis factor-α, interleukin (IL)-1β and IL-6, with the levels reaching its highest value at 10 µg/ml. IL-6 and IL-1β expression increased with increasing LPS concentration. These findings suggest that LPS reduced HK-2 cell viability whilst increasing the expression of inflammatory factors. Following transfection with ATM shRNA, expression levels of key autophagy indicators microtubule associated protein 1 light chain 3α I/II ratio and beclin-1 in the two ATM shRNA groups were also significantly reduced compared with the NC shRNA group. In summary, downregulation of ATM expression in HK-2 cells reduced LPS-induced inflammation and autophagy in sepsis-induced AKI in vitro, suggesting that LPS may induce autophagy in HK-2 cells through the ATM pathway leading to the upregulation of inflammatory factors.

Krüppel‑like factor4 ameliorates diabetic kidney disease by activating autophagy via the mTOR pathway.

Diabetic kidney disease (DKD) is diagnosed increasingly frequently and represents a serious threat to human health. Krüppel-like factor 4 (KLF4) has aroused attention due to its potential effect on podocytes and in ameliorating proteinuria associated with glomerulopathy. The purpose of the present study was to investigate the potential role of KLF4 in DKD. It was hypothesized that KLF4 impacts diabetic nephropathy by mediating the podocyte autophagic process. A KLF4 plasmid vector was constructed, and podocytes were transfected and incubated with DKD mice serum for in vitro experiments. A db/db spontaneous DKD mouse model was also established for in vivo study. After treatment, the level of serum creatinine (Scr), blood urea nitrogen (BUN), and 24-h urinary protein was determined. Immunofluorescence and periodic acid-Schiff staining, western blotting, flow cytometry and a TUNEL assay were performed to observe changes in glomerular morphology and the level of apoptosis, cytoskeleton proteins, epithelial-mesenchymal transition (EMT) biomarkers, autophagic proteins and mTOR pathway proteins in each group. KLF4 overexpression significantly reduced the level of urinary albumin, Scr, BUN and attenuated mesangial matrix expansion, as well as mesangial cell proliferation in DKD mice. KLF4 overexpression also inhibited podocyte apoptosis and downregulated vimentin and α-smooth muscle actin, and upregulated E-cadherin and nephrin, both in vivo and in vitro. Moreover, the microtubule associated protein 1 light chain 3α (LC3)-II/LC3-I ratio and LC3-II fluorescence was significantly increased in the vector-KLF4 group compared to the negative control vector group both in vivo and in vitro. Finally, a decrease in the level of phosphorylated (p)-mTOR and p-S6K protein expression was observed following KLF4 overexpression in vitro. The present findings suggested that KLF4 plays a renoprotective role in DKD, which is associated with the activation of podocyte autophagy, and may be involved in the mTOR signaling pathway.

Quercetin has a protective effect on atherosclerosis via enhancement of autophagy in ApoE-/- mice.

The present study examined the involvement of autophagy as a mechanism in the protective effect of quercetin (QUE) on atherosclerosis (AS) in ApoE−/− mice. An AS model was established by feeding ApoE−/− mice a high-fat diet (HFD). Mice were divided into four experimental groups: The model, QUE, 3-methyladenine (3-MA) and QUE + 3-MA groups. Additionally, age-matched wild-type C57BL/6 mice were used as a Control group. Autophagosomes in the aorta were examined using a transmission electron microscope. Aorta pathology, serum lipid accumulation and collagen deposition were determined by hematoxylin and eosin, Oil Red O and Masson staining, respectively. The levels of cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-18 (IL-18) were measured using ELISA assays. Protein levels of mTOR, microtubule associated protein 1 light chain 3a (LC3), P53 and cyclin dependent kinase inhibitor 1A (P21) in the aorta were analyzed using western blotting. ApoE−/− mice which were fed HFD exhibited substantial AS pathology, no autophagosomes, higher levels of TNF-α, IL-1β, IL-18 and mTOR and lower ratios of LC3 II/I. All these alterations were ameliorated and aggravated by QUE and 3-MA treatment, respectively. The inhibition of AS by QUE may be associated with the enhancement of autophagy and upregulation of P21 and P53 expression.