LC3B Protein Research Articles

Inhibitory Effect of Ursolic Acid on Proliferation and Migration of Renal Carcinoma Cells and Its Mechanism.

Background Renal carcinoma is one of the most common malignant tumors in the urinary system. Autophagy can be both activated and inhibited in renal carcinoma, and it plays a double-edged role in the development of renal carcinoma. In the early stage of cancer, autophagy can suppress tumors. In the late stage, autophagy contributes to the survival of tumor cells in an unfavorable environment, and some autophagy-related proteins P62, LC3B, and beclin-1 have become indicators of the prognosis of patients with renal carcinoma. Aim To demonstrate that ursolic acid activates autophagy in renal carcinoma 786-O cells by inhibiting the hedgehog signaling pathway. Methods The effect of ursolic acid on the viability of 786-O cells was determined by the MTT method; the effect of ursolic acid on the proliferation and migration of 786-O cells was examined by crystalline violet staining and scratch assay, respectively. For the study of autophagy, we firstly screened the time points. Western blot assay was used to detect the expression level of autophagic protein P62 at different time points of ursolic acid on 786-O. Then, the Cell MeterTM Autophagy Assay Kit was used to detect the effect of different doses of ursolic acid on the autophagic fluorescence intensity of 786-O cells; the Western blot method was used to detect the effect of different doses of ursolic acid on the expression levels of LC3II and P62 proteins in 786-O cells. Further, AdPlus-mCherry-GFP-LC3B adenovirus transfection was used to detect the effect of ursolic acid on the autophagic flow of 786-O cells; ursolic acid was combined with the autophagy inhibitor chloroquine (CQ) to detect the expression level of autophagy protein LC3II by Western blot. In terms of mechanism, the effect of ursolic acid on hedgehog signaling pathway-related proteins in 786-O cells was detected by Western blot. Results Ursolic acid inhibited the activity, proliferation, and migration of 786-O cells, enhanced the fluorescence intensity of autophagosomes in 786-O cells, increased the expression level of autophagy marker protein LC3II, and inhibited the expression level of P62 in a time and dose-dependent manner; ursolic acid activated the autophagic flow in 786-O cells, which showed that ursolic acid caused the accumulation of autophagic fluorescent spots and enhanced the fluorescence intensity of autophagosomes. Ursolic acid activated the autophagic flow in 786-O cells, as evidenced by the accumulation of autophagic fluorescent spots and enhanced fluorescence intensity of autophagosomes, and the combined use of the autophagy inhibitor CQ increased the expression level of LC3II compared to ursolic acid alone; ursolic acid decreased the expression levels of PTCH1, GLI1, SMO, SHH, and c-Myc and increased the expression level of Sufu in the hedgehog signaling pathway. Conclusion Ursolic acid activates autophagy in renal carcinoma 786-O cells, probably by inhibiting hedgehog signaling pathway activity.

TGFβ Induces Changes in Mitochondrial Homeostasis in Skeletal Muscle of Mice with Bone Metastases

Cancer cachexia is a common neoplastic syndrome of advanced cancers characterized by a loss of skeletal muscle mass and decreased function. Patients with cancer cachexia have higher chances of morbidity and mortality. We have shown that mice inoculated with human MDA‐MB‐231 breast cancer cells, which metastasize to bone, have skeletal muscle weakness, cachexia, and altered skeletal muscle metabolism. Mice with bone metastases have increased serum levels of transforming growth factor beta (TGFβ). Serum TGFβ decreased after treatment with the bone anti‐resorptive agent, zoledronic acid (ZA), suggesting TGFβ is, in part, bone‐derived. Additionally, mice with bone metastases treated with ZA had increased skeletal muscle function. TGFβ has also been shown to decrease the master regulator of mitochondrial biogenesis, peroxisome‐activated receptor γ coactivator 1α (PGC1α), in C2C12 myotubes. Mitochondria provide muscle with energy (ATP) via oxidative phosphorylation, so we measured oxygen consumption and energy production using indirect calorimetry in mice with bone metastases. Oxygen consumption and energy production were both decreased in these mice suggesting a decrease in mitochondrial function. We then examined skeletal muscle mitochondria using transmission electron microscopy and western blotting. We found highly dysmorphic mitochondria and altered protein markers of mitochondrial biogenesis and dynamics. Additionally, LC3B protein was increased in skeletal muscle mitochondria demonstrating increased mitophagy in skeletal muscle of mice with bone metastases. From our previous knowledge and data of TGFβ and its role in mice with bone metastases, we hypothesize that TGFβ causes mitochondrial dysfunction in skeletal muscle of mice with bone metastases. We have shown that a 48‐hour treatment with TGFβ decreases protein expression in markers of mitochondrial biogenesis and dynamics in C2C12 myotubes. This data suggests that TGFβ could be causing the mitochondrial dysfunction and dysmorphia we see in our mouse model, but further experiments are needed to prove this hypothesis. Our data demonstrates the importance of mitochondrial function in skeletal muscle cells and skeletal muscle of mice with bone metastases. Understanding the role TGFβ plays in changes of skeletal muscle mitochondria in mice with bone metastases will help contribute to defining treatments for patients with cancer cachexia.

Mother with Obesity Induced by High‐Fat Diet Impaired Autophagic Process and Induced Renal Lipid Accumulation in the Offspring

IntroductionThe prevalence of obesity has increased worldwide, and more women are entering obese pregnancy. The offspring born to maternal obesity have an increased risk of obesity and insulin resistance in later life and could affect offspring’s kidney injury. The abnormal renal lipid metabolism leads to renal lipid accumulation and these suggested to be the importance mechanism to develop and progress of chronic kidney disease (CKD). Lipophagy is the autophagic process to hydrolyze lipid droplet into free fatty acid for preserve energy and prevent an excess of lipid accumulation. The defect in lipophagy regulation is associated with renal lipid accumulation and contribute to kidney injury. Therefore, we hypothesized that maternal obesity induced by high‐fat diet programmed to increase offspring renal lipid accumulation via dysregulation of autophagic process and abnormal renal lipid metabolism leading to offspring’s kidney injury.MethodsFemale C57BL/6 mice were divided into two groups, control lean mother and maternal obesity groups, fed with standard and high‐fat diet, respectively, for 8 weeks. After that, female mice were mated with normal male mice and were housed and maintain each diet throughout gestation and lactation periods. After weaning, male offspring from each mother were sacrificed and blood samples and kidney were collected to evaluate autophagic and lipid metabolism.ResultsMaternal obesity was significantly increased body weight, fat weight, blood glucose and HOMA‐IR in the offspring. The increasing in renal cholesterol and triglyceride together with upregulated FAS, Srebp1, ADFP and CD36 protein expression indicating increased renal lipid synthesis and renal lipid uptake were observed in the offspring born to maternal obesity. Meanwhile, maternal obesity affected to downregulated PPARα and CPT1A demonstrating decreased renal lipolysis leading to exacerbated renal lipid accumulation in the offspring born to maternal obesity. The significant increase in mTOR, decrease in AMPK, Beclin‐1, LC3B and LAMP2 protein expression were shown in offspring’s kidney born to maternal obesity. These represented the inhibition of autophagic process, impaired autophagosome formation along with aberrant lysosome formation.ConclusionMaternal obesity affected to program offspring obese‐insulin resistance and increased the risk of kidney injury. The stimulation of renal lipogenesis and the suppression of renal lipolysis led to increased renal lipid accumulation. Moreover, maternal obesity induced impaired autophagic process by decreasing autophagosome formation and impaired lysosome formation. These alterations reduced the hydrolyzation of lipid droplet in the offspring’s kidney leading to more severity of renal lipid accumulation and resulting in offspring’s kidney injury.

Agomelatine Ameliorates Obesity‐Induced Kidney Injury through the Inhibition of Renal Fibrosis and Improvement of Impaired Autophagy

IntroductionChronic kidney disease is the crucial complication that could occur in obesity. Prolong high‐fat diet consumption can stimulate reactive oxygen species (ROS) and contribute to kidney injury through various pathways. Autophagy plays an important role for maintaining intracellular homeostasis. ROS could induce impaired autophagy, subsequently led to kidney damage. Agomelatine (AGOM), a melatonin analogue, is indicated for the treatment of major depressive disorders. The antioxidant and anti‐inflammation effects have been reported in AGOM treatment in chronic mild stress‐induced depression. Previous study found that AGOM inhibited apoptosis and ER stress in the kidney under high‐fat diet condition in rats. However, the effect of AGOM supplementation on renal fibrosis and impaired autophagy in obese condition has never been elucidated.AimsTo evaluate the renoprotective effect of AGOM on kidney function in high‐fat diet‐induced kidney injury, as well as to explore the underlying mechanisms involving autophagy signaling pathway.Materials and methodsMale Wistar rats were received normal diet (ND) or high‐fat diet (HF) for 16 weeks. Then, the HF rats were separated into 4 subgroups including (1) HF; (2) agomelatine20 (AGOM20); (3) AGOM40, the rats were received AGOM at the dose of 20 and 40 mg/kg/day, respectively; and (4) N‐acetylcysteine (NAC), the rats were received NAC at the dose of 100 mg/kg/day by oral gavage for 4 weeks. After 4 weeks of treatment, all rats were sacrificed. Blood, urine and kidney tissues were collected for further investigations.ResultsThe results demonstrated that HF rats developed kidney dysfunction as shown by the raising in serum creatinine, creatinine clearance along with the changes of glomerular structure and the elevating of positive area of α‐SMA, which is renal fibrosis indicator. These alterations were reversed by AGOM and NAC treatment. Moreover, HF rats showed renal oxidative stress as indicated by the increasing in PKCα and NOX4 expression and decreasing in antioxidant enzyme, GCLC. AGOM and NAC administrations reduced oxidative stress and restored antioxidant enzyme. Furthermore, high‐fat diet consumption markedly reduced AMPK, along with the elevation of mTOR expression. Additionally, we found that HF rats aggravated impaired autophagy which could induce oxidative stress, fibrosis and finally led to kidney cell death. AGOM administration restored autophagy process as shown by the increasing in Beclin‐1, LC3B and Atg5 protein. These results demonstrated that AGOM improved kidney injury through the regulation of oxidative stress, fibrosis and autophagy process.ConclusionsThese findings indicated that AGOM prevented kidney injury under obese condition via the inhibition of oxidative stress and fibrosis and improved impaired autophagy via regulating AMPK‐mTOR‐autophagy signaling pathways.

LC3 subfamily in cardiolipin-mediated mitophagy: a comparison of the LC3A, LC3B and LC3C homologs

ABSTRACT Externalization of the phospholipid cardiolipin (CL) to the outer mitochondrial membrane has been proposed to act as a mitophagy trigger. CL would act as a signal for binding the LC3 macroautophagy/autophagy proteins. As yet, the behavior of the LC3-subfamily members has not been directly compared in a detailed way. In the present contribution, an analysis of LC3A, LC3B and LC3C interaction with CL-containing model membranes, and of their ability to translocate to mitochondria, is described. Binding of LC3A to CL was stronger than that of LC3B; both proteins showed a similar ability to colocalize with mitochondria upon induction of CL externalization in SH-SY5Y cells. Besides, the double silencing of LC3A and LC3B proteins was seen to decrease CCCP-induced mitophagy. Residues 14 and 18 located in the N-terminal region of LC3A were shown to be important for its recognition of damaged mitochondria during rotenone- or CCCP-induced mitophagy. Moreover, the in vitro results suggested a possible role of LC3A, but not of LC3B, in oxidized-CL recognition as a counterweight to excessive apoptosis activation. In the case of LC3C, even if this protein showed a stronger CL binding than LC3B or LC3A, the interaction was less specific, and colocalization of LC3C with mitochondria was not rotenone dependent. These results suggest that, at variance with LC3A, LC3C does not participate in cargo recognition during CL-mediated-mitophagy. The data support the notion that the various LC3-subfamily members might play different roles during autophagy initiation, identifying LC3A as a novel stakeholder in CL-mediated mitophagy.Abbreviations: ACTB/β-actin: actin beta; Atg8: autophagy-related 8; CL: cardiolipin; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; DMSO: dimethyl sulfoxide; DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DTT: DL-dithiothreitol; FKBP8: FKBP prolyl isomerase 8; GABARAP: GABA type A receptor associated protein; GABARAPL1: GABA type A receptor associated protein like 1; GABARAPL2: GABA type A receptor associated protein like 2; GFP: green fluorescent protein; IMM: inner mitochondrial membrane; LUV/LUVs: large unilamellar vesicle/s; MAP1LC3A/LC3A: microtubule associated protein 1 light chain 3 alpha; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAP1LC3C/LC3C: microtubule associated protein 1 light chain 3 gamma; NME4/NDPK-D/Nm23-H4: NME/NM23 nucleoside diphosphate kinase 4; O/A: oligomycin A + antimycin A; OMM: outer mitochondrial membrane; PA: phosphatidic acid; PC: phosphatidylcholine; PG: phosphatidylglycerol; PINK1: PTEN induced putative kinase 1; PtdIns4P: phosphatidylinositol-4-phosphate; Rho-PE: lissamine rhodamine phosphatidylethanolamine; SUV/SUVs: small unilamellar vesicle/s

Low doses of niclosamide and quinacrine combination yields synergistic effect in melanoma via activating autophagy-mediated p53-dependent apoptosis

Malignant melanoma is a highly aggressive, malignant, and drug-resistant tumor. It lacks an efficient treatment approach. In this study, we developed a novel anti-melanoma strategy by using anti-tapeworm drug niclosamide and anti-malarial drug quinacrine, and investigated the molecular mechanism by in vitro and in vivo assays. Meanwhile, other types of tumor cells, immortalized epithelial cells and bone marrow mesenchymal stem cells were used to evaluate the universal role of anti-cancer and safety of the strategy. The results showed, briefly, an exposure to niclosamide and quinacrine led to an increased apoptosis-related protein p53, cleaved caspase-3 and cleaved PARP and autophagy-related protein LC3B expression, and a decreased expression of autophagy-related protein p62, finally leading to cell apoptosis and autophage. After inhibiting autophagy by Baf-A1, flow cytometry and western blot showed that the expression of apoptosis-related proteins was down-regulated and the number of apoptotic cells decreased. Subsequently, in the siRNA-mediated p53 knockdown cells, the expression of apoptosis-related proteins and the number of apoptotic cells were also reduced, while the expression of autophagy-related proteins including LC3B, p62 did not change significantly. To sum up, we developed a new, safe strategy for melanoma treatment by using low doses of niclosamide and quinacrine to treat melanoma; and found a novel mechanism by which the combination application of low doses of niclosamide and quinacrine exerts an efficient anti-melanoma effect through activation of autophagy-mediated p53-dependent apoptosis. The novel strategy was verified to exert a universal anti-cancer role in other types of cancer.

Brugia malayi Microfilariae Induce Autophagy through an Interferon-γ Dependent Mechanism in Human Monocytes.

Monocyte dysfunction in helminth infection is one of the mechanisms proposed to explain the diminished parasite antigen-specific T cell responses seen with patent filarial infection. In fact, monocytes from filariae-infected individuals demonstrate internalized filarial antigens and, as a consequence, express inhibitory surface molecules and have diminished cytokine production. To investigate the mechanisms underlying monocyte dysfunction in filarial infections, purified human monocytes were exposed to live microfilariae (mf) of Brugia malayi, and the mRNA and protein expression of important inhibitory and/or autophagy-related molecules were assessed. Our data indicate that mf-induced autophagy in human monocytes shown by the formation of autophagic vesicles, by the upregulation in the mRNA expression of autophagy-related genes BCN1, LC3B, ATG5, ATG7 (P < 0.05), and by increase in the levels of LC3B protein. Furthermore, this mf-induced autophagy increased the levels of monocyte CD206 expression. In addition, mf significantly induced the frequency of interferon (IFN)-γ+ human monocytes and at the same time induced the mRNA expression of indoleamine 2,3-dioxygenase (IDO) through an IFN-γ-dependent mechanism; significantly enhanced tryptophan degradation (an indicator of IDO activity; P < 0.005). Interestingly, this autophagy induction by mf in monocytes was IFN-γ-dependent but IDO-independent as was reversed by anti-IFN-γ but not by an IDO inhibitor. Our data collectively suggest that mf of Brugia malayi regulate the function of monocytes by induction of IDO and IFN-γ, induce autophagy through an IFN-γ-dependent mechanism, and increase M2 phenotype through induction of autophagy; all acting in concert to drive monocyte dysfunction.

Neuroprotective effect of liraglutide in an experimental mouse model of multiple sclerosis: role of AMPK/SIRT1 signaling and NLRP3 inflammasome

The heterogeneous nature of multiple sclerosis (MS) and the unavailability of treatments addressing its intricate network and reversing the disease state is yet an area that needs to be elucidated. Liraglutide, a glucagon-like peptide-1 analogue, recently exhibited intriguing potential neuroprotective effects. The currents study investigated its potential effect against mouse model of MS and the possible underlying mechanisms. Demyelination was induced in C57Bl/6 mice by cuprizone (400 mg/kg/day p.o.) for 5 weeks. Animals received either liraglutide (25 nmol/kg/day i.p.) or dorsomorphin, an AMPK inhibitor, (2.5 mg/Kg i.p.) 30 min before the liraglutide dose, for 4 weeks (starting from the second week). Liraglutide improved the behavioral profile in cuprizone-treated mice. Furthermore, it induced the re-myelination process through stimulating oligodendrocyte progenitor cells differentiation via Olig2 transcription activation, reflected by increased myelin basic protein and myelinated nerve fiber percentage. Liraglutide elevated the protein content of p-AMPK and SIRT1, in addition to the autophagy proteins Beclin-1 and LC3B. Liraglutide halted cellular damage as manifested by reduced HMGB1 protein and consequently TLR-4 downregulation, coupled with a decrease in NF-κB. Liraglutide also suppressed NLRP3 transcription. Dorsomorphin pre-administration indicated a possible interplay between AMPK/SIRT1 and NLRP3 inflammasome activation as it partially reversed liraglutide’s effects. Immunohistochemical examination of Iba+ microglia emphasized these findings. In conclusion, liraglutide exerts neuroprotection against cuprizone-induced demyelination via anti-inflammatory, autophagic flux activation, NLRP3 inflammasome suppression, and anti-apoptotic mechanisms, possibly mediated, at least in part, via AMPK/SIRT1, autophagy, TLR-4/ NF-κB/NLRP3 signaling.Graphical abstractThe potential mechanistic insight of Lira in alleviating Cup-induced neurotoxicity via: (1) AMPK/SIRT1 pathways activation resulting in the stimulation of brain autophagy flux (confirmed by lowering Beclin-1 and LC3-B protein expression). (2) Inhibition of NLRP3 inflammasome activation, as evidenced by reduced HMGB1, TLR-4, NF-κB and NLRP3 protein expression, alongside diminishing the activation of its downstream cascade as reflected by reduced levels of caspase-1 and IL-1β protein expression. (3) A possible modulating interplay between the previously mentioned two pathways.

Asthma Susceptibility Gene ORMDL3 Promotes Autophagy in Human Bronchial Epithelium.

The genome-wide association study (GWAS)-identified asthma susceptibility risk alleles on chromosome 17q21 increase the expression of ORMDL3 (ORMDL sphingolipid biosynthesis regulator 3) in lung tissue. Given the importance of epithelial integrity in asthma, we hypothesized that ORMDL3 directly impacted bronchial epithelial function. To determine whether and how ORMDL3 expression impacts the bronchial epithelium, in studies using both primary human bronchial epithelial cells and human bronchial epithelial cell line, 16HBE (16HBE14o-), we assessed the impact of ORMDL3 on autophagy. Studies included: autophagosome detection by electron microscopy, RFP-GFP-LC3B to assess autophagic activity, and Western blot analysis of autophagy-related proteins. Mechanistic assessments included immunoprecipitation assays, intracellular calcium mobilization assessments, and cell viability assays. Coexpression of ORMDL3 and autophagy-related genes was measured in primary human bronchial epithelial cells derived from 44 subjects. Overexpressing ORMDL3 demonstrated increased numbers of autophagosomes and increased levels of autophagy-related proteins LC3B, ATG3, ATG7, and ATG16L1. ORMDL3 overexpression promotes autophagy and subsequent cell death by impairing intracellular calcium mobilization through interacting with SERCA2. Strong correlation was observed between expression of ORMDL3 and autophagy-related genes in patient-derived bronchial epithelial cells. Increased ORMDL3 expression induces autophagy, possibly through interacting with SERCA2, thereby inhibiting intracellular calcium influx, and induces cell death, impairing bronchial epithelial function in asthma.

5-Bromo-3,4-dihydroxybenzaldehyde Promotes Hair Growth through Activation of Wnt/β-Catenin and Autophagy Pathways and Inhibition of TGF-β Pathways in Dermal Papilla Cells.

Various studies addressing the increasing problem of hair loss, using natural products with few side effects, have been conducted. 5-bromo-3,4-dihydroxybenzaldehyde (BDB) exhibited anti-inflammatory effects in mouse models of atopic dermatitis and inhibited UVB-induced oxidative stress in keratinocytes. Here, we investigated its stimulating effect and the underlying mechanism of action on hair growth using rat vibrissa follicles and dermal papilla cells (DPCs), required for the regulation of hair cycle and length. BDB increased the length of hair fibers in rat vibrissa follicles and the proliferation of DPCs, along with causing changes in the levels of cell cycle-related proteins. We investigated whether BDB could trigger anagen-activating signaling pathways, such as the Wnt/β-catenin pathway and autophagy in DPCs. BDB induces activation of the Wnt/β-catenin pathway through the phosphorylation of GSG3β and β-catenin. BDB increased the levels of autophagic vacuoles and autophagy regulatory proteins Atg7, Atg5, Atg16L, and LC3B. We also investigated whether BDB inhibits the TGF-β pathway, which promotes transition to the catagen phase. BDB inhibited the phosphorylation of Smad2 induced by TGF-β1. Thus, BDB can promote hair growth by modulating anagen signaling by activating Wnt/β-catenin and autophagy pathways and inhibiting the TGF-β pathway in DPCs.

Role of Glucocorticoid Signaling and HDAC4 Activation in Diaphragm and Gastrocnemius Proteolytic Activity in Septic Rats.

Sepsis increases glucocorticoid and decreases IGF-1, leading to skeletal muscle wasting and cachexia. Muscle atrophy mainly takes place in locomotor muscles rather than in respiratory ones. Our study aimed to elucidate the mechanism responsible for this difference in muscle proteolysis, focusing on local inflammation and IGF-1 as well as on their glucocorticoid response and HDAC4-myogenin activation. Sepsis was induced in adult male rats by lipopolysaccharide (LPS) injection (10 mg/kg), and 24 h afterwards, rats were euthanized. LPS increased TNFα and IL-10 expression in both muscles studied, the diaphragm and gastrocnemius, whereas IL-6 and SOCS3 mRNA increased only in diaphragm. In comparison with gastrocnemius, diaphragm showed a lower increase in proteolytic marker expression (atrogin-1 and LC3b) and in LC3b protein lipidation after LPS administration. LPS increased the expression of glucocorticoid induced factors, KLF15 and REDD1, and decreased that of IGF-1 in gastrocnemius but not in the diaphragm. In addition, an increase in HDAC4 and myogenin expression was induced by LPS in gastrocnemius, but not in the diaphragm. In conclusion, the lower activation of both glucocorticoid signaling and HDAC4-myogenin pathways by sepsis can be one of the causes of lower sepsis-induced proteolysis in the diaphragm compared to gastrocnemius.

Combined Effects of Fludarabine and Interferon Alpha on Autophagy Regulation Define the Phase of Cell Survival and Promotes Responses in LLC-MK2 and K562 Cells.

Autophagy is a known mechanism of cells under internal stress that regulates cellular function via internal protein recycling and the cleaning up of debris, leading to healthy live cells. However, the stimulation of autophagy by external factors such as chemical compounds or viral infection mostly tends to induce apoptosis/cell death. This study hypothesizes that manipulation of the autophagy mechanism to the pro-cell survival and/or decreased pro-viral niche can be a strategy for effective antiviral and anticancer treatment. Cells susceptible to viral infection, namely LLC-MK2, normal monkey epithelium, and K562, human immune-related lymphocyte, which is also a cancer cell line, were treated with fludarabine nucleoside analog (Fdb), interferon alpha (IFN-α), and a combination of Fdb and IFN-α, and then were evaluated for signs of adaptive autophagy and STAT1 antiviral signaling by Western blotting and immunolabeling assays. The results showed that the low concentration of Fdb was able to activate an autophagy response in both cell types, as demonstrated by the intense immunostaining of LC3B foci in the autophagosomes of living cells. Treatment with IFN-α (10 U/mL) showed no alteration in the initiator of mTOR autophagy but dramatically increased the intracellular STAT1 signaling molecules in both cell types. Although in the combined Fdb and IFN-α treatment, both LLC-MK2 and K562 cells showed only slight changes in the autophagy-responsive proteins p-mTOR and LC3B, an adaptive autophagy event was clearly shown in the autophagosome of the LLC-MK2 cell, suggesting the survival phase of the normal cell. The combined effect of Fdb and IFN-α treatment on the antiviral response was identified by the level of activation of the STAT1 antiviral marker. Significantly, the adaptive autophagy mediated by Fdb was able to suppress the IFN-α-mediated pSTAT1 signaling in both cell types to a level that is appropriate for cellular function. It is concluded that the administration of an appropriate dose of Fdb and IFN-α in combination is beneficial for the treatment of some types of cancer and viral infection.

Alterations in hippocampal mitochondrial dynamics areassociated with neurodegeneration and recognition memory decline in old male mice.

Mitochondrial dynamics is a key process that modulates the ultrastructure, quality and function of mitochondria. It is disrupted in numerous major neurodegenerative disorders including Parkinson's, Alzheimer's and Huntington's disease. Mitochondrial dysfunction has been correlated with the loss of memory. Previous studies suggest the involvement of Vdac1 and Drp1 in outer mitochondrial membrane permeabilization and promotion of mitochondrial fragmentation through Drp1 phosphorylation at S616. However, alterations in mitochondrial dynamics with respect to aging, memory loss and neurodegeneration remain unexplored. Therefore, the present study focuses on the involvement of mitochondrial dynamics in neurodegeneration and recognition memory decline during aging. The recognition memory decline was validated by the novel object recognition test and measurement of hippocampal Arc protein level during aging. The ultrastructure analysis revealed a decline in mitochondrial length and area, while an increase in the number of fragmented, round and disrupted mitochondria in the hippocampus during aging. Disruption was also evident in mitochondrial cristae and membrane with advancing age. The change in mitochondrial morphology was corroborated by an increase in the expression of phospho-Drp1 (S616) and Cyt-c proteins but decline in Mfn2, LC3B, Vdac1, Bcl-XL and Bcl-2 proteins in the hippocampus during aging. Taken together, our findings reveal that an increase in the expression of phospho-Drp1 (S616) and decrease in Mfn2 and LC3B proteins in the hippocampus bring about a reduction in mitochondrial length and area, and rise in mitochondrial fragmentation leading to reduced neuronal cell density, increased neurodegeneration and recognition memory decline in old male mice. Diagram depicts the increase in hippocampal mitochondrial fragmentation during aging of mice. Increased mitochondrial fragmentation causes distorted mitochondrial function such as decrease in ATP/ADP transportation due to decrease in Vdac1 protein level and increase in oxidative damage. These alterations result in hippocampal neurodegeneration and consequently impairment in recognition memory during aging.

Raloxifene prevents intracellular invasion of pathogenic bacteria through modulation of cell metabolic pathways.

Pathogenic bacteria can invade and survive inside phagocytic and non-phagocytic cells and use them as a sanctuary against antibiotics. Induction of various host protective mechanisms, including autophagy, can be a novel and effective method to combat intracellular bacteria. Recent studies report that raloxifene, a selective oestrogen receptor modulator, can induce cellular autophagy. To demonstrate the effect of raloxifene on intracellular invasion and proliferation of pathogenic bacteria and investigate raloxifene-induced metabolic changes in host cells. Autophagic induction was determined by the extent of nuclear fragmentation and expression levels of the LC3B protein. Intracellular invasion of MRSA strains into A549 lung epithelial cells and invasion of Mycobacterium abscessus into RAW264.7 macrophages were assessed by invasion and proliferation assays. Changes in host cell metabolism were examined by transcriptomic profiling using RNA sequencing. Our data demonstrate increased autophagy in cells upon raloxifene treatment, which contributed to prevention of bacterial invasion and proliferation. Transcriptomic profiling of host cells revealed changes in cholesterol-related pathways and consequent increases in oxidative stress-related genes and in autophagic induction through the TRIM and GABA pathways. Our results demonstrate raloxifene's potential as a broad-spectrum antibacterial agent through autophagic induction in host cells and prevention of intracellular invasion and proliferation of pathogenic bacteria.

Autophagy-Mediated Synaptic Refinement and Auditory Neural Pruning Contribute to Ribbon Synaptic Maturity in the Developing Cochlea.

In rodents, massive initial synapses are formed in the auditory peripheral nervous system at the early postnatal stage, and one of the major phenomena is that the number of afferent synapses in the cochlea is significantly reduced in the duration of development. This raises the hypothesis that the number of cochlear ribbon synapses are dramatically changed with hearing development and maturation. In this study, several tracers identifying activities of autophagy were applied to estimate the level of autophagy activity in the process of ribbon synapse development in mice; further, changes in the synaptic number and spiral ganglion nerve (SGN) fibers were quantitatively measured. We found robust expression of LC3B and lysosomal-associated membrane protein 1 as well as LysoTracker in or near inner hair cells and cochlear ribbon synapses in the early stage of postnatal development. Moreover, we found a significant loss in the intensity of SGN fibers at ribbon synaptic development and hearing onset. Thus, this study demonstrates that ribbon synaptic refinement and SGN fibers pruning are closely associated with the morphological and functional maturation of ribbon synapses and that synaptic refinement and SGN fiber pruning are regulated by the robust activities of autophagy in the earlier stages of auditory development.

Changes of gut microbiota in diabetic nephropathy and its effect on the progression of kidney injury.

We aimed to illustrate gut microbiota and short chain fatty acid (SCFA) levels in diabetic nephropathy (DN) patients, and investigate the mechanism of sodium butyrate in diabetic mellitus (DM) rats. Gut microbiota and serum SCFA levels were measured by 16S rDNA and GC-MS. After being built by streptozotocin (DM rats), the DM rats were administered 300 mg/kg sodium butyrate for 12 weeks (DM + BU rats). Gut microbiota, serum and fecal butyrate level were measured. RT-PCR, WB and transmission electron microscopy were performed to explore LC3mRNA or LC3B protein expression, and autophagosomes in kidney tissues. AMPK/mTOR protein expression in renal tissue were also measured. The gut microbial dysbiosis was found in DM and DN groups, and some SCFAs-producing bacteria were decreased in DN group. The serum butyrate concentrations were lower in SCFA-DN group compared with SCFA-HC group and SCFA-DM group in the other cohort. Serum butyrate level was positively correlated with eGFR. Sodium butyrate increased serum and fecal butyrate levels, and improved the enlargement of glomerular area and fibronectin and collagen IV expressions in renal tissues in DM + BU rats. The LC3 mRNA, LC3BII/I ratio and number of autophagosomes were increased in renal tissue of DM + BU rats. Higher p-AMPK/AMPK ratio and lower p-mTOR/ mTOR ratio were shown in renal tissue of DM + BU rats compared with DM rats. We found the decrease in SCFAs-producing bacteria and low SCFAs concentrations in DN patients. Oral butyrate supplementation may improve kidney injury in DM rats, possibly by increasing autophagy via activating AMPK/mTOR pathway.

Involvement of NEAT1/PINK1-mediated mitophagy in chronic obstructive pulmonary disease induced by cigarette smoke or PM2.5.

BackgroundThis study sought to explore the underlying mechanism of long non-coding ribonucleic acid nuclear enriched abundant transcript 1 (NEAT1) and PTEN-induced kinase 1 (PINK1)-mediated mitophagy in chronic obstructive pulmonary disease (COPD) induced by cigarette smoke (CS) or fine particular matter (PM2.5).MethodsIn total, 30 male Wistar Rats were divided into the following 3 groups: (I) the COPD group exposed to CS (CSM); (II) the COPD group exposed to PM2.5 (PMM); and (III) the control (Ctrl) group. Pulmonary function, the enzyme-linked immunoassay analysis results, the histopathology results, and the ultrastructures of the lung tissues were examined in the 3 groups, and NEAT1 expression levels and the mitophagy-related protein PINK1, Parkin, LC3B, and p62 levels were assessed by quantitative reverse transcription PCR (RT-qPCR) and Western blotting. The A549 cells were transfected with small interfering ribonucleic acid (siRNA) targeting NEAT1, and subsequently stimulated with CS extract (CSE) and PM2.5 suspension (PMS). Mitochondrial dysfunction and enhanced mitophagy were observed, and the expression of the NEAT1/PINK1 pathway was assessed by RT-qPCR and Western blotting.ResultsBoth the CSM and PMM groups had a lower tidal volume (VT), minute ventilation (MV), and a higher respiratory rate (f) than the Ctrl group. The interleukin (IL)-6, IL-8, and tumor necrosis factor–alpha levels in the serum and bronchoalveolar lavage fluid of the CSM and PMM groups were significantly increased. The histological examination results revealed airway remodeling, the formation of pulmonary bullae, and emphysema in the CSM and PMM groups. Subsequently, the ultrastructures of the lung tissues in the CSM and PMM groups showed mitochondrial swelling and autophagosomes. Additionally, NEAT1 expression, the level of the mitophagy-related protein PINK1, Parkin, and the ratio of LC3-II/I increased synchronously. Further, NEAT1 siRNA blocked PINK1 expression, inhibited mitochondrial dysfunctions, and mitophagy activation in the A549 cells exposed to CSE or PMS.ConclusionsOur results suggest that CS and PM2.5 exposure induce mitochondrial dysfunction, and the NEAT1/PINK1 pathway plays a critical role in the occurrence and development of COPD by regulating mitophagy.

Plasmodium Circumsporozoite Protein Enhances the Efficacy of Gefitinib in Lung Adenocarcinoma Cells by Inhibiting Autophagy via Proteasomal Degradation of LC3B.

Background: Almost all lung adenocarcinoma (LUAD) patients with EGFR mutant will develop resistance to EGFR-TKIs, which limit the long-term clinical application of these agents. Accumulating evidence shows one of the main reasons for resistance to EGFR-TKIs is induction of autophagy in tumor cells. Our previous study found that circumsporozoite protein (CSP) in Plasmodium can suppress autophagy in host hepatocytes. However, it is unknown whether CSP-mediated inhibition of autophagy could improve the anti-tumor effect of EGFR-TKIs. Methods: We constructed A549 and H1975 cell lines with stable overexpression of CSP (OE-CSP cells). CCK-8, Lactate Dehydrogenase (LDH), flow cytometry, and colony analysis were performed to observe the effect of CSP overexpression on cell viability, apoptosis rate, and colony formation ratio. The sensitizing effect of CSP on gefitinib was evaluated in vivo using a subcutaneous tumor model in nude mice and immunohistochemical assay. The role of CSP in regulation of autophagy was investigated by laser confocal microscopy assay and western blotting. A transcriptome sequencing assay and real-time polymerase chain reaction were used to determine the levels of mRNA for autophagy-related proteins. Cycloheximide (CHX), MG132, TAK-243, and immunoprecipitation assays were used to detect and confirm proteasomal degradation of LC3B. Results: OE-CSP A549 and H1975 cells were more sensitive to gefitinib, demonstrating significant amounts of apoptosis and decreased viability. In the OE-CSP group, autophagy was significantly inhibited, and there was a decrease in LC3B protein after exposure to gefitinib. Cell viability and colony formed ability were recovered when OE-CSP cells were exposed to rapamycin. In nude mice with xenografts of LUAD cells, inhibition of autophagy by CSP resulted in suppression of cell growth, and more marked apoptosis during exposure to gefitinib. CSP promoted ubiquitin-proteasome degradation of LC3B, leading to inhibition of autophagy in LUAD cells after treatment with gefitinib. When LUAD cells were treated with ubiquitin activating enzyme inhibitor TAK-243, cell viability, apoptosis, and growth were comparable between the OE-CSP group and a control group both in vivo and in vitro. Conclusion: CSP can inhibit gefitinib-induced autophagy via proteasomal degradation of LC3B, which suggests that CSP could be used as an autophagy inhibitor to sensitize EGFR-TKIs.

Corresponding Changes of Autophagy-Related Genes and Proteins in Different Stages of Knee Osteoarthritis: An Animal Model Study.

ObjectiveTo investigate the effect of autophagy expression levels of different weight‐bearing states and different stages of osteoarthritis in animal models, as well as the corresponding mechanisms.MethodsWe used the male Sprague–Dawley (SD) rats (12‐week‐old, SPF) to establish the OA animal models by modified Hulth method, and grouped animal models according to the length of time after surgery and different weight‐bearing areas. RT‐qPCR was carried out for detection of autophagy‐related genes such as Atg7, Atg12, P62, etc. Western blot analysis was used to detect the expression levels of corresponding autophagy‐related proteins such as LC3B, P62, etc. T test was performed for statistical analysis to compare different groups, while the differences were deemed statistically significant with P < 0.05. Transmission electron microscopy was used to observe the autophagosome to demonstrate the level of autophagy expression and the status of the chondrocytes.ResultsThe results of the RT‐qPCR testing showed that when the weight‐bearing cartilage of the 4‐week group (relatively mild) was compared with that of the 10‐week group (relatively severe), there were statistically significant differences in all the genes tested, in detail: Atg3 (P < 0.01), Atg7 (P < 0.01), Atg12 (P < 0.01), P62 (P < 0.0001). The expression of autophagy‐related mRNA in the 4‐week group is increased compared with that of the 10‐week group. As for the expression of proteins, Western blotting showed that in the comparison between the 4‐ and the 10‐week groups, statistically significant results include Atg12 (P < 0.01) in the non‐weight‐bearing area, with decreased autophagy in the 10‐week group compared with that of the 4‐week group, while expression of LC3B (P < 0.05) protein was significantly higher in the 4‐week group than in the control in the non‐weight‐bearing area. The expression of LC3B (P < 0.0001) and P62 (P < 0.05) in the 10‐week group were higher than that of the control. Transmission electron microscope showed that autophagy in the weight‐bearing area is stronger than that in the non‐weight‐bearing area, and autophagy in the 4‐week group is stronger than in the 10‐week group for the weight‐bearing area.ConclusionsThe expression of autophagy varies during different stages of osteoarthritis, in which the autophagy is stronger in the early stage of osteoarthritis, and gradually decreases with the progression of the disease. Autophagy in different weight‐bearing areas may also be different.

Belimumab Decreases Autophagy and Citrullination in Peripheral Blood Mononuclear Cells from Patients with Systemic Lupus Erythematosus.

Belimumab (BLM) is a B lymphocyte stimulator (BLyS) inhibitor approved for the treatment of systemic lupus erythematosus (SLE). Autophagy is a cell survival mechanism involved in the pathogenesis of SLE. Citrullination is a post-translational modification catalyzed by peptidylarginine deiminase (PAD) enzymes. Autophagy and citrullination may generate neoepitopes, evoking an autoimmune response. No previous studies have investigated the connection of these processes, and how BLM could affect them, in SLE. Ex vivo autophagy and protein citrullination were analyzed by western blot in lysates from 26 SLE patients’ PBMCs at baseline and after 2, 4, and 12 weeks of BLM administration, and from 16 healthy donors’ PBMCs. Autophagic PBMCs were identified by the immunofluorescent detection of the autophagy-associated proteins LC3B (LC3 puncta) and LAMP-1. Autophagosome accumulation was evaluated in CD14− (PBLs) and CD14+ (monocytes) SLE cells. The presence of the BLyS receptors BAFF-R, BCMA, and TACI on SLE CD4+, CD8+ T cells and monocytes, as well as serum IL-18 levels, was also assessed. Following BLM administration, we observed a decrease in autophagy and citrullination, with a lowering of LC3-II, citrullinated vimentin, and PAD4 expression levels in PBMCs from SLE patients. LC3-II levels showed a correlation with the SLE Disease Activity Index 2000 (SLEDAI-2K) after 12 weeks of therapy. The LC3B/LAMP-1 analysis confirmed the reduction in autophagy. A lesser autophagosome accumulation occurred in PBLs and monocytes which, in turn, seemed to be the main cellular populations contributing to autophagy. A reduction in patients’ serum IL-18 concentrations occurred. CD4+ and CD8+ cells weakly expressed BAFF receptors; monocytes expressed only BAFF-R. BLM could impact on autophagy and citrullination, offering an opportunity for a deeper understanding of these mechanisms in SLE, and a possible tool for the clinical management of SLE.