OBJECTIVE: To investigate whether autophagy mediates the effects of aldehyde dehydrogenase 2 (ALDH2) on the proliferation of neonatal rat cardiac fibroblasts cultured in high glucose. METHODS: Cardiac fibroblasts were isolated from neonatal (within 3 days) SD rats and subcultured. The fibroblasts of the third passage, after identification with immunofluorescence staining for vimentin, were treated with 5.5 mmol/L glucose (control group), 30 mmol/L glucose (high glucose group), or 30 mmol/L glucose in the presence of Alda-1 (an ALDH2 agonist), daidzin (an ALDH2 2 inhibitor), or both. Western blotting was employed to detect ALDH2, microtubule-associated protein 1 light chain 3B subunit (LC3B) and Beclin-1 in the cells, and a hydroxyproline detection kit was used for determining hydroxyproline content in cell culture medium; CCK- 8 kit was used for assessing the proliferation ability of the cardiac fibroblasts after the treatments. RESULTS: Compared with the control cells, the cells exposed to high glucose exhibited obviously decreased expressions of ALDH2, Beclin-1 and LC3B and increased cell number and hydroxyproline content in the culture medium. Treatment of the high glucose-exposed cells with Alda-1 significantly increased Beclin-1, LC3B, and ALDH2 protein expressions and lowered the cell number and intracellular hydroxyproline content, whereas the application of daidzin resulted in reverse changes in the expressions of ALDH2, Beclin-1 and LC3B, viable cell number and intracellular hydroxyproline content in high glucose-exposed cells. CONCLUSIONS Mitochondrial ALDH2 inhibits the proliferation of neonatal rat cardiac fibroblasts induced by high glucose, and the effect is possibly mediated by the up-regulation of autophagy-related proteins Beclin-1 and LC3B.
Aldehyde Dehydrogenase ; Aldehyde Dehydrogenase, Mitochondrial ; metabolism ; Animals ; Animals, Newborn ; Autophagy ; Beclin-1 ; physiology ; Fibroblasts ; Glucose ; Microtubule-Associated Proteins ; Mitochondrial Proteins ; Rats ; Rats, Sprague-Dawley

We have previously reported that Cystatin C (CysC) is a pivotal mediator in the neuroprotection induced by hyperbaric oxygen (HBO) preconditioning; however, the underlying mechanism and how CysC changes after stroke are not clear. In the present study, we demonstrated that CysC expression was elevated as early as 3 h after reperfusion, and this was further enhanced by HBO preconditioning. Concurrently, LC3-II and Beclin-1, two positive-markers for autophagy induction, exhibited increases similar to CysC, while knockdown of CysC blocked these elevations. As a marker of autophagy inhibition, p62 was downregulated by HBO preconditioning and this was blocked by CysC knockdown. Besides, the beneficial effects of preserving lysosomal membrane integrity and enhancing autolysosome formation induced by HBO preconditioning were abolished in CysC rats. Furthermore, we demonstrated that exogenous CysC reduced the neurological deficits and infarct volume after brain ischemic injury, while 3-methyladenine partially reversed this neuroprotection. In the present study, we showed that CysC is biochemically and morphologically essential for promoting autophagic flux, and highlighted the translational potential of HBO preconditioning and CysC for stroke treatment.
Animals ; Autophagy ; physiology ; Beclin-1 ; metabolism ; Brain ; metabolism ; pathology ; Brain Ischemia ; metabolism ; pathology ; therapy ; Cystatin C ; genetics ; metabolism ; Disease Models, Animal ; Gene Expression ; Gene Knockdown Techniques ; Hyperbaric Oxygenation ; Lysosomes ; metabolism ; pathology ; Male ; Microtubule-Associated Proteins ; metabolism ; Neurons ; metabolism ; pathology ; Neuroprotection ; physiology ; Oxygen ; therapeutic use ; Random Allocation ; Rats, Sprague-Dawley ; Rats, Transgenic ; Reperfusion Injury ; metabolism ; pathology ; therapy

Autophagy is an evolutionarily-conserved self-degradative process that maintains cellular homeostasis by eliminating protein aggregates and damaged organelles. Recently, vesicle-associated membrane protein-associated protein B (VAPB), which is associated with the familial form of amyotrophic lateral sclerosis, has been shown to regulate autophagy. In the present study, we demonstrated that knockdown of VAPB induced the up-regulation of beclin 1 expression, which promoted LC3 (microtubule-associated protein light chain 3) conversion and the formation of LC3 puncta, whereas overexpression of VAPB inhibited these processes. The regulation of beclin 1 by VAPB was at the transcriptional level. Moreover, knockdown of VAPB increased autophagic flux, which promoted the degradation of the autophagy substrate p62 and neurodegenerative disease proteins. Our study provides evidence that the regulation of autophagy by VAPB is associated with the autophagy-initiating factor beclin 1.
Autophagy ; drug effects ; physiology ; Beclin-1 ; genetics ; metabolism ; Cell Line, Transformed ; Gene Expression Regulation ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Microtubule-Associated Proteins ; genetics ; metabolism ; R-SNARE Proteins ; genetics ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; metabolism ; RNA-Binding Proteins ; genetics ; metabolism ; Transfection

A growing body of evidence has indicated the important role of autophagy receptors in directing ubiquitinated or non-ubiquitinated cargos towards autophagy. Autophagy receptors bind to LC3 (microtubule-associated protein 1 light chain 3) on phagophore and autophagosome membranes, and recognize signals on cargoes in the delivery system of autophagy. However, the diverse domains in the receptor structures determine that their roles would never be limited to autophagy. Up to date, increasing numbers of the receptor proteins have been demonstrated to serve as a molecular link or switch participating in autophagic degradation, apoptosis or cell survival signals. Here, we highlight the non-autophagic roles of these receptor proteins to draw attention to this growing research topic.
Apoptosis ; Autophagy ; Humans ; Microtubule-Associated Proteins ; physiology ; Signal Transduction ; Ubiquitination

To observe the effect of high-mobility group box 1 (HMGB1) on autophagy and chemotherapy resistance in human leukemiacell line (K562) cells, and to explore the underlying mechanisms.
 Methods: The K562 cells were cultured in vitro and divided into 6 groups: a chemotherapeutic group, a chemotherapeutic control group, a HMGB1 preconditioning group, a HMGB1 preconditioning control group, a HMGB1 siRNA group and a siRNA control group. The chemotherapeutic group was further divided into a vincristine (VCR) group, an etoposide (VP-16) group, a cytosine arabinoside (Ara-C) group, a adriamycin (ADM) group and a arsenic trioxide (As2O3) group. The cell activity was evaluated by cell counting kit-8. The protein levels of HMGB1, microtubule-associate protein1light chain3 (LC3), AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (m-TOR) were determined by Western blotting. The level of serum HMGB1 was evaluated by enzyme-linked immunosorbent assay (ELISA). The autophagy was examined by monodansylcadaverine staining and observed under transmission electron microscopy.
 Results: Compared with the control group, the cell activity was significantly decreased and the level of serum HMGB1 was significantly increased in the chemotherapeutic (VCR, VP-16, Ara-C, ADM and As2O3) groups (all P<0.05). Compared with the control group, the cell activity and the level of serum HMGB1 were significantly increased in the HMGB1 preconditioning group (both P<0.05). Compared with the siRNA control group, the cell activity and the level of serum HMGB1 were significantly decreased in the HMGB1 siRNA group (both P<0.05). Compared with the control group, the expression of LC3-II and the formation of autophagic bodies were increased in the HMGB1 preconditioning group (both P<0.05), the p-AMPK expression was increased and p-mTOR expression was decreased (both P<0.05).
 Conclusion: HMGB1 can increase the autophagy and promote chemotherapy resistance through the pathway of AMPK/m-TOR in K562 cells.
AMP-Activated Protein Kinases ; genetics ; physiology ; Arsenic Trioxide ; Arsenicals ; Autophagy ; genetics ; Cytarabine ; Doxorubicin ; Drug Resistance, Neoplasm ; genetics ; physiology ; Etoposide ; HMGB1 Protein ; genetics ; physiology ; Humans ; K562 Cells ; physiology ; Microtubule-Associated Proteins ; Oxides ; RNA, Small Interfering ; Signal Transduction ; TOR Serine-Threonine Kinases ; genetics ; physiology ; Vincristine

BACKGROUNDAutophagy has been found to be involved in animal and cell models of atherosclerosis, but to date, it lacks general observation in human atherosclerotic plaques. Here, we investigated autophagy in smooth muscle cells (SMCs), endothelial cells (ECs), and macrophages in human atherosclerotic plaques via transmission electron microscopy (TEM), western blotting, and immunohistochemistry analysis.

METHODSThe histopathologic morphology of these plaques was observed via hematoxylin and eosin staining. The ultrastructural morphology of the SMCs, ECs, and macrophages in these plaques was observed via TEM. The localization of microtubule-associated protein 1 light chain 3 (MAP1-LC3), a relatively special maker of autophagy, in plaques was observed by double fluorescent immunochemistry and western blotting.

RESULTSAll of these human atherosclerotic plaques were considered advanced and unstable in histologically observation. By double fluorescent immunochemistry, the expression of LC3-II increased in the SMCs of the fibrous cap, the macrophages, and the microvascular ECs of the plaque shoulders. The protein level of LC3-II by western blotting significantly increased in plaques compared with normal controls. In addition, TEM observation of plaques revealed certain features of autophagy in SMCs, ECs, and macrophages including the formation of myelin figures, vacuolization, and the accumulation of inclusions in the cytosol. These results indicate that autophagy is activated in SMCs, ECs, and macrophages in human advanced atherosclerotic plaques.

CONCLUSIONSOur study is to demonstrate the existence of autophagy in human atherosclerotic plaques by different methods, which may contribute to the development of pharmacological approaches to stabilize vulnerable and rupture-prone lesions.

Atherosclerosis ; metabolism ; physiopathology ; Autophagy ; physiology ; Endothelial Cells ; pathology ; Humans ; In Vitro Techniques ; Microscopy, Electron, Transmission ; Microtubule-Associated Proteins ; metabolism ; Myocytes, Smooth Muscle ; pathology ; Plaque, Atherosclerotic ; metabolism ; physiopathology ; ultrastructure

In this study, we determined how rosiglitazone (RSG) differentially affected hippocampal neurogenesis in mice fed a low-fat diet (LFD) or high-fat diet (HFD; 60% fat). LFD and HFD were given to the mice for 8 weeks. Four weeks after initiating the LFD and HFD feeding, vehicle or RSG was administered orally once a day to both groups of mice. We measured cell proliferation and neuroblast differentiation in the subgranular zone of the dentate gyrus using Ki67 and doublecortin (DCX), respectively, as markers. In addition, we monitored the effects of RSG on the levels of DCX and brain-derived neurotrophic factor (BDNF) in hippocampal homogenates. At 8 weeks after the LFD feeding, the numbers of Ki67- and DCX-positive cells as well as hippocampal levels of DCX and BDNF were significantly decreased in the RSG-treated group compared to the vehicle-treated animals. In contrast, the numbers of Ki67- and DCX-positive cells along with hippocampal levels of DCX and BDNF in the HFD fed mice were significantly increased in the RSG-treated mice compared to the vehicle-treated group. Our data demonstrate that RSG can modulate the levels of BDNF, which could play a pivotal role in cell proliferation and neuroblast differentiation in the hippocampal dentate gyrus.
Animals ; Blotting, Western ; Brain-Derived Neurotrophic Factor/metabolism ; Cell Differentiation/*drug effects ; Cell Proliferation/drug effects ; Dentate Gyrus/growth & development/physiology ; Diet, Fat-Restricted ; *Diet, High-Fat ; Hippocampus/growth & development/physiology ; Hypoglycemic Agents/*pharmacology ; Immunohistochemistry ; Ki-67 Antigen/metabolism ; Male ; Mice, Inbred C57BL ; Microtubule-Associated Proteins/metabolism ; Neurogenesis/*drug effects ; Neuropeptides/metabolism ; Thiazolidinediones/*pharmacology

OBJECTIVEAutophagy is involved in several neurodegenerative diseases and recently its role in acute brain injury has won increasing interest. Spinal cord injuries (SCIs) often lead to permanent neurological deficit. Therefore, in this study, we examined the pro?les of autophagy-linked proteins (MAP-LC3) after SCI to investigate whether the expression of autophagy contributes to neurological deficit after SCI.

METHODSAdult female Sprague-Dawley rats were used and randomly divided into control and SCI groups. All the rates received laminectomy at T8-T10 level. Those in the SCI group received additional exposure of the dorsal surface of the spinal cord, followed by a weight- drop injury. Thereafter we investigated the expression levels of MAP-LC3, beclin-1, Cathepsin D and the beclin-1-binding protein bcl-2 by western blot analysis at 12 h, 24 h, 3 d, 7 d, 21 d and 28 d. One-way ANOVA with Tukey post hoc test was used to compare data between groups.

RESULTSWe observed significant increase in the level of LC3 (LC3-II/LC3-I) at 3 d and 7 d after SCI when compared with the sham group. While the level of beclin-1 and ratio of beclin-1/bcl-2 was found to have increased from 12 h to 24 h after injury. Cathepsin D expression was also elevated at 7 d (P<0.01).

CONCLUSIONBased on the above mentioned data, we proposed that autophagy plays a role in the manifestation of cell injury following SCI.

Adaptor Proteins, Signal Transducing ; metabolism ; Animals ; Apoptosis Regulatory Proteins ; metabolism ; Autophagy ; physiology ; Beclin-1 ; Blotting, Western ; Cathepsin D ; metabolism ; Disease Models, Animal ; Female ; Laminectomy ; Microtubule-Associated Proteins ; metabolism ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries ; metabolism

OBJECTIVETo study the changes in microtubule motor protein expression in the spinal cord and sciatic nerve of rats exposed to carbon disulfide, and to investigate the possible molecular mechanism of changes in axonal transport in carbon disulfide-induced peripheral neuropathy.

METHODSHealthy adult male Wistar rats were randomly divided into one control group and three experimental groups (10 rats per group). The rats in experimental groups were intoxicated by gavage of carbon disulfide at a dose of 200, 400, or 600 mg/kg 6 times a week for 6 consecutive weeks, while the rats in control group were given the same volume of corn oil by gavage. Animals were sacrificed after exposure, with nerve tissue separated. The levels of dynein, dynactin, and kinesin in the spinal cord and sciatic nerve were determined by Western blot.

RESULTSThe content of dynein, dynactin, and kinesin in the sciatic nerve decreased significantly under exposure to carbon disulfide. The levels of dynein in the sciatic nerve were reduced by 23.47% and 33.34% at exposure doses of 400 and 600 mg/kg, respectively. The levels of dynactin in the sciatic nerve of the three experimental groups were reduced by 19.91%, 24.23%, and 41.30%, respectively. The level of kinesin was reduced by 25.98%under exposure to 600 mg/kg carbon disulfide. All the differences were statistically significant (P < 0.01). As compared with the control group, the 600 mg/kg group experienced a 28.24% decrease in level of dynactin in the spinal cord (P < 0.01), but no significant change was observed in the level of dynein or kinesin.

CONCLUSIONCarbon disulfide has an impact on microtubule motor protein expression in nerve tissues, which might be involved in the development of carbon disulfide-induced peripheral neuropathy.

Animals ; Axonal Transport ; drug effects ; physiology ; Carbon Disulfide ; toxicity ; Dynactin Complex ; Male ; Microtubule-Associated Proteins ; metabolism ; Nerve Tissue ; metabolism ; Peripheral Nervous System Diseases ; chemically induced ; metabolism ; Rats, Wistar ; Sciatic Nerve ; metabolism ; Spinal Cord ; metabolism

OBJECTIVETo explore the effects and molecular mechanisms of rhein on reducing starvation-induced autophagic protein expression in renal tubular epithelial ( NRK-52E) cells.

METHODHank's balanced salt solution (HBSS) was used to induce NRK-52E cells to be in the state of starvation. After the intervention of HBSS for 0, 0.5,1, 2 and 6 hours, firstly, the protein expression of microtubule-associated protein 1 light chain 3(LC3 I/II), which is a key protein in autophagy, was detected. Secondly, the protein expressions of mammalian target of rapamycin (mTOR) and phosphorylated-mTOR Ser2448 (p-mTOR S2448) were examined. And then, after the co-treatment of rhein (5 mg x L(-1)) and HBSS (1 mL) without or with mTOR inhibitor, rapamycin (100 nmol x L(-1)), the protein expressions of LC3 I/II, mTOR and p-mTOR S2448 were tested, respectively.

RESULTHBSS could induce the up-regulation of LC3 II and the down-regulation of p-mTOR S2448 at protein expression level in NRK-52E cells. The co-treatment of rhein and HBSS could reversely regulate the protein expressions of LC3 II and p-mTOR S2448 in NRK-52E cells significantly. The co-treatment of rapamycin, rhein and HBSS could recover the level of LC3 II protein expression in HBSS-intervened NRK-52E cells.

CONCLUSIONHBSS induces autophagy in renal tubular epithelial cells by inhibiting mTOR signaling pathway activation. Rhein reduces the autophagic protein expression in renal tubular epithelial cells through regulating mTOR signaling pathway activation, which is the possible effects and molecular mechanisms.

Animals ; Anthraquinones ; pharmacology ; Autophagy ; drug effects ; Cells, Cultured ; Epithelial Cells ; drug effects ; metabolism ; Isotonic Solutions ; pharmacology ; Kidney Tubules ; drug effects ; metabolism ; Microtubule-Associated Proteins ; genetics ; Rats ; Signal Transduction ; drug effects ; TOR Serine-Threonine Kinases ; antagonists & inhibitors ; genetics ; physiology