OBJECTIVE: To investigate whether hydrogen-rich water exerts a protective effect against cellular injury by affecting the level of autophagy after oxygen glucose deprivation/reoxygenation (OGD/R) in a mouse hippocampal neuronal cell line (HT22 cells). METHODS: HT22 cells in logarithmic growth phase were cultured in vitro. Cell viability was detected by cell counting kit-8 (CCK-8) assay to find the optimal concentration of Na₂S₂O₄. HT22 cells were divided into control group (NC group), OGD/R group (sugar-free medium+10 mmol/L Na₂S₂O₄ treated for 90 minutes and then changed to normal medium for 4 hours) and hydrogen-rich water treatment group (HW group, sugar-free medium+10 mmol/L Na₂S₂O₄ treated for 90 minutes and then changed to medium containing hydrogen-rich water for 4 hours). The morphology of HT22 cells was observed by inverted microscopy; cell activity was detected by CCK-8 method; cell ultrastructure was observed by transmission electron microscopy; the expression of microtubule-associated protein 1 light chain 3 (LC3) and Beclin-1 was detected by immunofluorescence; the protein expression of LC3II/I and Beclin-1, markers of cellular autophagy, was detected by Western blotting. RESULTS: Inverted microscopy showed that compared with the NC group, the OGD/R group had poor cell status, swollen cytosol, visible cell lysis fragments and significantly lower cell activity [(49.1±2.7)% vs. (100.0±9.7)%, P < 0.01]; compared with the OGD/R group, the HW group had improved cell status and remarkably higher cell activity [(63.3±1.8)% vs. (49.1±2.7)%, P < 0.01]. Transmission electron microscopy showed that the neuronal nuclear membrane of cells in the OGD/R group was lysed and a higher number of autophagic lysosomes were visible compared with the NC group; compared with the OGD/R group, the neuronal damage of cells in the HW group was reduced and the number of autophagic lysosomes was notably decreased. The results of immunofluorescence assay showed that the expressions of LC3 and Beclin-1 were outstandingly enhanced in the OGD/R group compared with the NC group, and the expressions of LC3 and Beclin-1 were markedly weakened in the HW group compared with the OGD/R group. Western blotting assay showed that the expressions were prominently higher in both LC3II/I and Beclin-1 in the OGD/R group compared with the NC group (LC3II/I: 1.44±0.05 vs. 0.37±0.03, Beclin-1/β-actin: 1.00±0.02 vs. 0.64±0.01, both P < 0.01); compared with the OGD/R group, the protein expression of both LC3II/I and Beclin-1 in the HW group cells were notably lower (LC3II/I: 0.54±0.02 vs. 1.44±0.05, Beclin-1/β-actin: 0.83±0.07 vs. 1.00±0.02, both P < 0.01). CONCLUSIONS Hydrogen-rich water has a significant protective effect on OGD/R-causing HT22 cell injury, and the mechanism may be related to the inhibition of autophagy.
Mice ; Animals ; Oxygen/metabolism* ; Beclin-1/pharmacology* ; Glucose/metabolism* ; Actins ; Sincalide ; Autophagy/physiology* ; Hydrogen/pharmacology* ; Reperfusion Injury ; Apoptosis

This study aimed to investigate the effects of hypoxia on RhoA/Rho-kinase (ROCK) signaling pathway and autophagy in pulmonary artery smooth muscle cells (PASMCs), and to explore the underlying mechanism of Umbelliferone (Umb) in ameliorating chronic hypoxic pulmonary hypertension. PASMCs were cultured from Sprague-Dawley (SD) rats and randomly divided into control group, hypoxia group, hypoxia + Umb intervention group and normoxia + Umb intervention group. Alpha smooth muscle actin (α-SMA) and LC3 were assessed by immunofluorescence staining. Protein expression of RhoA, ROCK2, p-MYPT1, LC3-II, Beclin-1, p62, C-Caspase 3, Bax and Bcl-2 was analyzed by Western blotting. In in vivo study, SD rats were divided into control group, hypoxia group and hypoxia + Umb intervention group. Weight ratio of the right ventricle (RV)/left ventricle plus septum (LV+S) was detected, and pulmonary arterial morphological features were examined by HE staining. The results indicated that compared with the control group, the LC3-II/LC3-I ratio and expression of Beclin-1 were significantly increased, while p62 expression was significantly decreased, and the expressions of RhoA, ROCK2 and p-MYPT1 were significantly increased in PASMCs of hypoxia group (P < 0.05). The changes of LC3-II/LC3-I ratio, the expressions of Beclin-1, p62, RhoA, ROCK2 and p-MYPT1 in PASMCs were reversed by Umb treatment (P < 0.05). Consistently, the pulmonary arterial wall was thickened and the RV/(LV+S) ratio was increased in hypoxic rats, which were significantly improved by Umb treatment (P < 0.05). These results suggest that Umb can improve hypoxia-induced pulmonary hypertension by inhibiting the RhoA/ROCK signaling pathway and autophagy in PASMCs.
Animals ; Autophagy ; Beclin-1/pharmacology* ; Hypertension, Pulmonary/etiology* ; Hypoxia/complications* ; Myocytes, Smooth Muscle/metabolism* ; Pulmonary Artery ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Umbelliferones/pharmacology* ; rho-Associated Kinases/pharmacology*

OBJECTIVES: To study the role and mechanism of autophagy in lipopolysaccharide (LPS)-induced inflammatory response of human alveolar epithelial A549 cells. METHODS: A549 cells were stimulated with LPS to establish a cell model of inflammatory response, and were then grouped (n=3 each) by concentration (0, 1, 5, and 10 μg/mL) and time (0, 4, 8, 12, and 24 hours). The A549 cells were treated with autophagy inhibitor 3-methyladenine (3-MA) to be divided into four groups (n=3 each): control, LPS, 3-MA, and 3-MA+LPS. The A549 cells were treated with autophagy agonist rapamycin (RAPA) to be divided into four groups (n=3 each): control, LPS, RAPA, and RAPA+LPS. The A549 cells were transfected with the Toll-like receptor 4 (TLR4) overexpression plasmid to be divided into four groups (n=3 each): TLR4 overexpression control, TLR4 overexpression, TLR4 overexpression control+LPS, and TLR4 overexpression+LPS. The A549 cells were transfected with TLR4 siRNA to be divided into four groups (n=3 each): TLR4 silencing control,TLR4 silencing, TLR4 silencing control+LPS, and TLR4 silencing+LPS. CCK-8 assay was used to measure cell viability. Western blot was used to measure the protein expression levels of inflammatory indicators (NLRP3, Caspase-1, and ASC), autophagic indicators (LC3B, Beclin-1, and P62), and TLR4. RESULTS: After stimulation with 1 μg/mL LPS for 12 hours, the levels of inflammatory indicators (NLRP3, Caspase-1, and ASC), autophagic indicators (LC3B, Beclin-1, and P62), and TLR4 increased and reached the peak (P<0.05). Compared with the LPS group, the 3-MA+LPS group had reduced expression of autophagy-related proteins and increased expression of inflammation-related proteins and TLR4, while the RAPA+LPS group had increased expression of autophagy-related proteins and reduced inflammation-related proteins and TLR4 (P<0.05). The TLR4 overexpression+LPS group had reduced autophagy-related proteins and increased inflammation-related proteins compared with the TLR4 overexpression control+LPS group, and the TLR4 silencing+LPS group had increased autophagy-related proteins and reduced inflammation-related proteins compared with the TLR4 silencing control+LPS group (P<0.05). CONCLUSIONS In the LPS-induced inflammatory response of human alveolar epithelial A549 cells, autophagic flux has a certain protective effect on A549 cells. TLR4-mediated autophagic flux negatively regulates the LPS-induced inflammatory response of A549 cells.
Humans ; A549 Cells ; Autophagy ; Beclin-1/metabolism* ; Caspase 1/metabolism* ; Inflammation ; Lipopolysaccharides/pharmacology* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Toll-Like Receptor 4/metabolism*

OBJECTIVETo explore the autophagy of RPMI8226 cells induced by As(2)O(3) and its possible mechanisms.

METHODSRPMI8226 was incubated with different concentration of As(2)O(3) for different time, and the inhibiting rate was calculated by MTT method. The autophagic rate of RPMI8226 cells incubated with different concentration of As(2)O(3) was determined by FACS. The change of cells ultrastructure was observed by transmission electron microsopy (TEM). After incubation with different concentration of As(2)O(3), the expression of Beclin-1 on RPMI8226 was detected by RT-PCR and Western blot.

RESULTSDifferent concentration of As(2)O(3) could significantly inhibit the proliferation of RPMI8226 cells (P < 0.05), and the inhibitory effect was in dose- and time-dependent way in a certain range. the autophagic rate increased with the increasing of drug concentration and prolonging of action time (P < 0.05). TEM results revealed a typical autophagosome in RPMI-8226 cell treated by As(2)O(3) for 48 hours. Beclin-1 was up-regulated in RPMI 8226 cells when treated with different concentration of As(2)O(3) for 48h (P < 0.05).

CONCLUSIONAs(2)O(3) can induce autophagy of RPMI8226 cells, and the mechanism may be associated with the upregulation of Beclin-1.

Apoptosis Regulatory Proteins ; metabolism ; Arsenicals ; pharmacology ; Autophagy ; drug effects ; Beclin-1 ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Membrane Proteins ; metabolism ; Oxides ; pharmacology ; Up-Regulation

To explore the mechanism of autophagic death of acute myelocytic leukemia cell U937 induced by clofarabine, the MTT bioassay was used to analyze the growth inhibitory effect and half inhibition concentration on U937 incubated in vitro with different concentrations of clofarabine for 24 and 48 hours, and the flow cytometry was used to detect the autophagy rate of U937. The expression of Beclin 1 in U937 treated by clofarabine for 48h was measured by Western blot. The results indicated that when U937 cells were treated with 0.01 µmol/L and 0.15 µmol/L clofarabine for 48 hours, the proliferation inhibition rate was 46.92% ± 4.24% and 86.10% ± 1.16%, and the half inhibition concentration of clofarabine was 0.022 µmol/L. With 0.01 µmol/L and 0.1 µmol/L clofarabine on U937 for 48 hours, the autophagy rate was 11.0033% ± 1.4387% and 59.4133% ± 3.5409%, and increased in dose-dependent manner (r = 0.99). Meanwhile the Beclin 1 was upregulated along with increase of clofarabine concentration, as compared with control group, the difference was statistically significant (P < 0.05). It is concluded that the different concentrations of clofarabine can significantly inhibit the proliferation of U937 in dose-dependent manner, and the mechanism of autophagic cell death in U937 may be associated with the upregulation of Beclin 1 expression.
Adenine Nucleotides ; pharmacology ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; metabolism ; Arabinonucleosides ; pharmacology ; Autophagy ; drug effects ; Beclin-1 ; Cell Proliferation ; drug effects ; Humans ; Membrane Proteins ; metabolism ; U937 Cells

This study aimed to explore the mechanism of Cistanches Herba in the treatment of cancer-induced fatigue(CRF) by network pharmacology combined with in vivo and in vitro experiments to provide a theoretical basis for the clinical medication. The chemical constituents and targets of Cistanches Herba were searched from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP). The targets of CRF were screened out by GeneCards and NCBI. The common targets of traditional Chinese medicine and disease were selected to construct a protein-protein interaction(PPI) network, followed by Gene Ontology(GO) functional and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analyses. A visual signal pathway rela-ted to Chinese medicine and disease targets was constructed. The CRF model was induced by paclitaxel(PTX) in mice. Mice were divided into a control group, a PTX model group, and low-and high-dose Cistanches Herba extract groups(250 and 500 mg·kg~(-1)). The anti-CRF effect in mice was evaluated by open field test, tail suspension test, and exhaustive swimming time, and the pathological morphology of skeletal muscle was evaluated by hematoxylin-eosin(HE) staining. The cancer cachexia model in C2C12 muscle cells was induced by C26 co-culture, and the cells were divided into a control group, a conditioned medium model group, and low-, medium-, and high-dose Cistanches Herba extract groups(62.5, 125, and 250 μg·mL~(-1)). The reactive oxygen species(ROS) content in each group was detected by flow cytometry, and the intracellular mitochondrial status was evaluated by transmission electron microscopy. The protein expression levels of hypoxia-inducible factor-1α(HIF-1α), BNIP3L, and Beclin-1 were detected by Western blot. Six effective constituents were screened out from Cistanches Herba. The core genes of Cistanches Herba in treating CRF were AKT1, IL-6, VEGFA, CASP3, JUN, EGFR, MYC, EGF, MAPK1, PTGS2, MMP9, IL-1B, FOS, and IL10, and the pathways related to CRF were AGE-RAGE and HIF-1α. Through GO enrichment analysis, it was found that the main biological functions involved were lipid peroxidation, nutrient deficiency, chemical stress, oxidative stress, oxygen content, and other biological processes. The results of the in vivo experiment showed that Cistanches Herba extract could significantly improve skeletal muscle atrophy in mice to relieve CRF. The in vitro experiment showed that Cistanches Herba extract could significantly reduce the content of intracellular ROS, the percentage of mitochondrial fragmentation, and the protein expression of Beclin-1 and increase the number of autophagosomes and the protein expression of HIF-1α and BNIP3L. Cistanches Herba showed a good anti-CRF effect, and its mechanism may be related to the key target proteins in the HIF-1α signaling pathway.
Animals ; Mice ; Cistanche ; Network Pharmacology ; Beclin-1 ; Reactive Oxygen Species ; Plant Extracts ; Drugs, Chinese Herbal/pharmacology* ; Molecular Docking Simulation ; Medicine, Chinese Traditional ; Neoplasms/genetics*

OBJECTIVE: To explore the effect of hnRNPK/Beclin1 signaling on the drug resistance of imatinib in Ph⁺ leukemia. METHODS: Expression level of hnRNPK was verified in the imatinib resistant and sensitive Ph⁺ leukemia cell lines by using Western blot. hnRNPK expression was down-regulated by using RNAi. Expression level of LC3I/II and Beclin1 were detected by Western blot and the sensitivity of imatinib was analyzed by CCK-8 assay before and after modulation of hnRNPK expression. RESULTS: hnRNPK showed overexpressed in imatinib resistant leukemia cell line. After the expression level of hnRNPK was down-regulated by RNAi, the sensitivity of drug resistance lines to imatinib restored, while the expression level of LC3I/II and Beclin1 were consistant with the modulation of hnRNPK expression. CONCLUSION hnRNP K/Beclin1 signaling may be involved in the development of imatinib resistance in Ph⁺ leukemia through the regulation of autophagy.
Antineoplastic Agents/pharmacology* ; Beclin-1 ; Cell Line, Tumor ; Drug Resistance ; Drug Resistance, Neoplasm ; Heterogeneous-Nuclear Ribonucleoprotein K ; Humans ; Imatinib Mesylate/pharmacology* ; Leukemia

OBJECTIVETo observe changes in the expression of autophagy-related proteins, Beclin-1 and LC3, in the hippocampal tissue of neonatal rats with hypoxic-ischemic brain damage (HIBD) at different time points, and to investigate the effect of rapamycin (Ra) on the expression of the above two proteins.

METHODSA total of 108 7-day-old Sprague-Dawley rats were randomly divided into sham, HIBD, and Ra groups (n=36 each). The HIBD model was established using the modified Rice method. For sham rats, only the left common carotid artery was separated without ligation or hypoxic treatment. For Ra-treated rats, 0.5 mg/kg Ra was administered by an intraperitoneal injection 1 hour before model establishment. The rats were anesthetized and sacrificed to collect brain tissues at 0, 6, 12, 24, 48, and 72 hours after model establishment. Changes in the expression of Beclin-1 and LC3 proteins in rat hippocampus were examined by Western blot.

RESULTSThe expression level of Beclin-1 in HIBD rats began to increase at 0 hour, peaked at 24 hours, and then declined thereafter, similar as those of Beclin-1 and LC3-II in Ra-treated rats. The expression level of LC3-II in HIBD rats began to increase at 0 hour, peaked at 12 hours, and then declined thereafter. At all time points, both Beclin-1 and LC3-II expression levels were significantly higher in HIBD and Ra-treated rats than in sham rats (P<0.05); except LC3-II at 12 hours, Beclin-1 and LC3-II expression levels were significantly higher in Ra-treated rats than in HIBD rats (P<0.05).

CONCLUSIONSHypoxia-ischemia activates autophagy in rat hippocampal cells, while Ra enhances the expression process of autophagy.

Animals ; Apoptosis Regulatory Proteins ; analysis ; Autophagy ; Beclin-1 ; Female ; Hippocampus ; chemistry ; Hypoxia-Ischemia, Brain ; metabolism ; Male ; Microtubule-Associated Proteins ; analysis ; Rats ; Rats, Sprague-Dawley ; Sirolimus ; pharmacology

OBJECTIVE: Background: We detect the effects of Beclinl on paclitaxel-sensitivity in laryngeal carcinoma cell. METHOD: This study used Hep-2, Hep-2-pcDNA3. 1, Hep-2-Beclinl as invitro model. The effect of paclitaxel on the proliferation and cell apoptosis of laryngeal cancer cell lines was evaluated by MTT assay and flow cytometry. The protein expression level of Akt and p-Akt was detected by Western blot. Result: After treated by paclitaxel, the inhibition rate was significantly higher in Hep-2-Beclin cells than in Hep-2-pcDNA3. 1 cells and Hep-2 cells (P<. 05). After dealing with 10 tg/L paclitaxel, the apoptosis rate in Hep-2, Hep-2-pcDNA3. 1, Hep-2-Beclinl were (23. 75 ± 2 3. 77) %, (21. 25 ± 4. 92) %, (32. 50 ± 5. 97) %, respectively. After dealing with 20µg/L paclitaxel, the apoptosis rate in Hep-2, Hep-2-pcDNA3. 1, Hep-2-Beclinl were (38. 75 ± 4. 79) %, (38. 75±6. 55) %, (50. 00±7. 26) %, respectively. Paclitaxel-induced apoptosis was higher in Hep-2-Beclin cells than in Hep-2-pcDNA3. 1 cells and Hep-2 cells (P<. 05). The result of western blot showed that the protein expression level of p-Akt in Hep-2-Beclin cells was lower than in Hep-2-pcDNA3. 1 cells and Hep-2 cells (P<0. 05) and the protein expression level of Akt was similar in three cell lines (P>0. 05). CONCLUSION Beclinl enhances paclitaxel-sensitivity by inhibition of PI3K/Akt pathway.
Apoptosis ; Apoptosis Regulatory Proteins ; physiology ; Beclin-1 ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Laryngeal Neoplasms ; pathology ; Larynx ; Membrane Proteins ; physiology ; Paclitaxel ; pharmacology ; Phosphatidylinositol 3-Kinases

OBJECTIVE: To study the molecular mechanism of PI3K-Ⅲ like functional domain inducing programmed cell death of leukemia cell line K562. METHODS: The purified PI3K-Ⅲ like functional domain protein was obtained by Pichia pastoris expression system. MTT assay and colony-forming assay were used to detect the effects of PI3K-Ⅲ like functional domain protein on K562 cell proliferation. The effects of PI3K-Ⅲ like functional domain protein on apoptosis and cell cycle of on K562 cells were detected by flow cytometry. The ultrastructural changes were detected by transmission electron microscopy. The expression of caspase-3 was detected by ELISA. The protein expressions of ATG4B, Beclin-1, Bcl-2 and LC3-II were evaluated by Western blot. RESULTS: PI3K-Ⅲ like functional domain protein could inhibit the proliferation and clony formation of K562 cells, which was significantly higher than the control group (P<0.05). In the experimental group, apoptosis and autophagosome were shown in K562 cells. The proportion of cells in G₀/G₁ phase increased significantly, while in S phase decreased significantly. Cell growth mostly stagnated in G₀/G₁ phase, which was significantly different from the control group (P<0.05). With the increase of concentration, the expression of caspase-3 protein increased significantly compared with the control group (r=0.966, P<0.05). The expression of ATG4B and beclin-1 appeared from increase to decrease, LC3-II increased while Bcl-2 decreased at different time points. CONCLUSION PI3K-Ⅲ like functional polypeptide could induce programmed cell death of leukemia cell K562. Beclin-1/Bcl-2 and caspase pathway may be involved in this way, which suggesting meant autophagy and apoptosis may work together at the same time.
Apoptosis ; Beclin-1/pharmacology* ; Caspase 3/metabolism* ; Cell Proliferation ; Humans ; K562 Cells ; Leukemia ; Phosphatidylinositol 3-Kinases/metabolism* ; Proto-Oncogene Proteins c-bcl-2/metabolism*