Objective: To explore the expression of autophagy related factors microtubule associated protein 1 light chain 3B (LC3B), p62, autophagy key factor Beclin1 in oral lichen planus (OLP) tissues and their relationships with the clinicopathological characteristics of OLP, investigating the function and significance of autophagy in pathogenesis of OLP. Methods: Forty-one lesion tissues (OLP group, twenty-one cases of erosive OLP and twenty cases of non-erosive OLP) were selected from OLP patients visiting the Department of Periodontal and Oral Medicine, School and Hospital of Stomatology, Guizhou Medical University from October 2017 to December 2019. Fifteen cases of normal oral mucosal tissues (control group) were collected from oral and maxillofacial surgery at The Affiliated Stomatology Hospital of Guizhou Medical University during the same period. Protein and mRNA expression levels of LC3B, p62 and Beclin1 were detected by immunohistochemistry (IHC) and real-time quantitative PCR (RT-qPCR) in OLP lesions respectively. The protein expression levels of LC3B, p62, Beclin1 and ratio of LC3B-Ⅱ/LC3B-Ⅰ in sixteen cases (eight cases of erosive OLP and eight cases of non-erosive OLP) from the OLP group were detected by Western blotting (WB). The potential relationship between LC3B, p62, Beclin1, LC3B-Ⅱ/LC3B-Ⅰ ratio and clinical features of OLP were analyzed. Results: IHC results showed that the positive expression rates of LC3B and p62 proteins in OLP lesion tissues [LC3B: 68% (28/41); p62: 59% (24/41)] were higher than those in the control group [LC3B: 5/15; p62: 3/15] (LC3B: χ²=5.55, P=0.019; p62: χ²=5.55, P=0.015). The positive expression rates of LC3B and p62 proteins in the erosive OLP group [LC3B: 86% (18/21); p62: 76% (16/21)] were higher than those in the non-erosive OLP group [LC3B: 50% (10/20); p62: 40% (8/20)] (LC3B: χ²=4.50, P=0.034; p62:χ²=5.53, P=0.019). The positive expression rate of Beclin1 protein in the OLP lesions[20% (8/41)] was lower than that in the control group (7/15) (χ²=4.13, P=0.042), but was not statistically different between the two types of OLP (P>0.05). The RT-qPCR results showed that the mRNA expression levels of LC3B and p62 in OLP lesions [LC3B: 2.78 (1.59, 6.15); p62: 4.30 (2.34, 6.29)] were higher than those in the control group [LC3B: 1.05 (0.88, 1.21); p62: 1.12 (0.89, 1.36)] (LC3B: Z=-4.56, P<0.001; p62: Z=-4.78, P<0.001), and the mRNA expression levels of LC3B and p62 in the erosive OLP group were higher than those in the non-erosive OLP group (LC3B: Z=-2.87, P=0.004; p62: Z=-2.95, P=0.003). The mRNA expression level of Beclin1 in OLP tissues was lower than that in the control group (Z=-2.43, P=0.015), but the difference was not statistically significant between the two types of OLP (P>0.05). WB results showed that the LC3B-Ⅱ/LC3B-Ⅰ ratio was higher in the OLP lesions than that in the control group (t=-2.45, P=0.021), and the LC3B-Ⅱ/LC3B-Ⅰ ratio was higher in the non-erosive OLP group than in the erosive OLP group (t=-2.38, P=0.032). Spearman's correlation analysis showed that the ratio was negatively correlated with the clinical staging and the degree of basal cell liquefaction in OLP (clinical staging: r=-0.57, P=0.021; basal cell liquefaction: r=-0.54, P=0.032), but not with the disease duration and the degree of lymphocytic infiltration (P>0.05). Conclusions: Autophagy related factors LC3B, p62 and Beclin1 may play a role in the formation and progression of OLP lesions. The autophagy level was relatively lack in erosive OLP compared to non-erosive OLP, contributing to the increased local lesion destruction in erosive OLP. Abnormal cellular autophagy may play an important role in the formation of OLP lesions.
Humans ; Lichen Planus, Oral/metabolism* ; Beclin-1 ; Microtubule-Associated Proteins/metabolism* ; Autophagy ; RNA, Messenger/metabolism*

OBJECTIVES: This work aimed to investigate the molecular mechanism of cyclic tensile stress (CTS) stimulating autophagy in human periodontal ligament cells (hPDLCs). METHODS: hPDLCs were isolated and cultured from normal periodontal tissues. hPDLCs were loaded with tensile stress by force four-point bending extender to simulate the autophagy of hPDLCs induced by orthodontic force du-ring orthodontic tooth movement. XMU-MP-1 was used to inhibit the Hippo signaling pathway to explore the role of the Hippo-YAP signaling pathway in activating hPDLC autophagy by tensile stress. The expression levels of autophagy-related genes (Beclin-1, LC3, and p62) in hPDLCs were detected by real-time quantitative polymerase chain reaction. Western blot was used to detect the expression levels of autophagy-related proteins (Beclin-1, LC3-Ⅱ/LC3-Ⅰ, and p62) and Hippo-YAP pathway proteins (active-YAP and p-YAP) in hPDLCs. Immunofluorescence was used to locate autophagy-related proteins (LC3-Ⅱand p62) and Hippo-YAP pathway proteins (active-YAP) of hPDLCs. RESULTS: CTS-activated autophagy in hPDLCs and expression of autophagy-related proteins initially increased and then decreased; it began to increase at 30 min, peaked at 3 h, and decreased (P<0.05). CTS increased the expression of active-YAP protein and decreased the expression of p-YAP protein (P<0.05). When XMU-MP-1 inhibited the Hippo-YAP signaling pathway (P<0.05), active-YAP protein was promoted to enter the nucleus and autophagy expression was enhanced (P<0.05). CONCLUSIONS The Hippo-YAP signaling pathway is involved in the regulation of autophagy activation in hPDLCs under CTS.
Humans ; Hippo Signaling Pathway ; Periodontal Ligament/metabolism* ; Beclin-1/metabolism* ; Cells, Cultured ; Autophagy

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

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*

To investigate the effect of miR-30a/HMGA2-mediated autophagy in osteosarcoma cells on apoptosis induced by chemotherapeutics. 
 Methods: A total of 30 osteosarcoma tissues of sensitive and resistant to chemotherapeutics were divided into a chemotherapy-sensitive group and a chemotherapy-resistant group. The mRNA expression levels of miR-30a and high mobility group protein A2 (HMGA2) in the chemotherapy-sensitive group and the chemotherapy-resistant group, and the mRNA expression levels of miR-30a in osteosarcoma U2-OS cells treated by cisplatin, doxorubicin and methotrexate at different concentrations were detected by real-time PCR. The expression levels of autophagy related protein Beclin 1, microtubule associated protein 1 light chain 3B (LC3B) and autophagy factor P62 were detected by Western blotting. The osteosarcoma U2-OS cells were transfected with miR-30a mimics and miR-30a inhibitors to construct a miR-30a high expression group, a miR-30a low expression group and a control group. The expression levels of Beclin 1, LC3B and P62 in osteosarcoma U2-OS cells after treatment of cisplatin and doxorubicin in these 3 groups were detected by Western blotting; the level of autophagy was detected by monodansylcada (MDC) staining; the level of ROS was detected by dihydroethidium (DHE); the level of cell surviving rate was detected by cell counting kit-8 (CCK-8); the level of apoptosis was detected by annexin APC/PI double staining; the level of mitochondria oxidative damage was detected by mitochondrial membrane potential assay kit with JC-1 (JC-1 method). The interaction between miR-30a and HMGA2 was detected by dual luciferase reporter assay. The osteosarcoma U2-OS cells were transfected with HMGA2 mimics and HMGA2-shRNA to construct a high HMGA2 group, a low HMGA2 group, and a control group. The expression levels of Beclin 1, LC3B and P62 in osteosarcoma U2-OS cells after the treatment of cisplatin were detected by Western blotting.
 Results: The level of miR-30a in the chemotherapy-resistant tissues was significantly lower than that in the chemotherapy-sensitive tissues (P<0.05), and the expression of HMGA2 was opposite comparing to that of miR-30a (P<0.05). After the treatment by low concentration (5 μmol/L) of chemotherapeutics, the level of miR-30a was down-regulated in osteosarcoma U2-OS cells, accompanied with up-regulation of Beclin 1 and LC3B (P<0.01) and down-regulation of P62 (P<0.01). Compared with the control group, the expression levels of Beclin 1 and LC3B were significantly decreased (P<0.05), and the expression level of P62 was significantly increased (P<0.05) in the miR-30a high expression group, which was opposite in the miR-30a low expression group. In the miR-30a high expression group treated by chemotherapeutics, the level of autophagy and the cell survival rate were lower than those in group with low expression of miR-30a, while the levels of ROS, the mitochondrial oxidative damage and the apoptosis were higher than those in group with low expression of miR-30a (all P<0.05). The targeting interaction between HMGA2 and miR-30a were verified by dual luciferase reporter assay. Compared with the control group, the expression levels of Beclin 1 and LC3B were significantly increased (P<0.05), and the expression level of P62 was significantly decreased (P<0.05) in the HMGA2 high expression group, which was opposite in the HMGA2 low expression group.
 Conclusion: Suppression of miR-30a/HMGA2-mediated autophagy in osteosarcoma cells is likely to enhance the therapeutic effect of chemotherapeutics.
Apoptosis ; Apoptosis Regulatory Proteins ; Autophagy ; Beclin-1 ; Bone Neoplasms ; Cell Line, Tumor ; HMGA2 Protein ; metabolism ; Humans ; MicroRNAs ; genetics ; Osteosarcoma

OBJECTIVE: To explore pro-oxidative state of rotator cuff tissue and expression levels of Beclin-1 and mam-malian target of rapamycin(mTOR) in patients with acute and chronic rotator cuff injury, and then analyzed relationship between rotator cuff injury and oxidative stress and autophagy. METHODS: Forty patients with rotator cuff injury were seleceted from July 2019 to December 2020, and divided into male chronic injury group, male acute injury group, female chronic injury group, and female acute injury group, 10 patients in each group. All patients were performed rotator cuff repair under arthroscopy. The sample of tendon at the rotator cuff injury site of the patient was taken during operation, and total reactive oxygen species (ROS) and superoxide dismutase(SOD) were detected by detection kit;expression of Beclin-1 and mTOR mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR), and Western-blot was applied to detect protein expression of Beclin-1 and p-mTOR/mTOR. RESULTS: There were no significant difference in expression of ROS, SOD, Beclin-1mRNA and mTOR mRNA between male and female chronic injury groups, and between male and female acute injury groups (P>0.05); ROS, SOD and Beclin-1mRNA in male chronic injury group were higher than those in male chronic injury group, while mTOR mRNAand protein decreased (P<0.05);ROS, SOD and Beclin-1 mRNA in female chronic injury group were up-regulated compared with female acute injury group, while mTOR mRNA was down-regulated (P<0.05). CONCLUSION Chronic rotator cuff injury is more likely to stimulate the pro-oxidation state of rotator cuff tissue than acute rotator cuff injury, which could up-regulating expression of autophagy factor Beclin-1 and down-regulating expression of mTOR. Therefore, patients with chronic rotator cuff injury may have higher levels of oxidative stress and autophagy.
Female ; Humans ; Male ; Beclin-1/metabolism* ; Reactive Oxygen Species/metabolism* ; RNA, Messenger/metabolism* ; Rotator Cuff/surgery* ; Rotator Cuff Injuries/surgery* ; Superoxide Dismutase/metabolism* ; TOR Serine-Threonine Kinases/metabolism*

OBJECTIVETo detect Beclin1 expression and explore its clinical significance in primary hepatocellular carcinoma (HCC).

METHODSBeclin1 expressions in 10 normal hepatic tissues, 30 hepatitis liver, 30 cirrhotic liver and 50 HCC tissues were detected by immunohistochemical staining.

RESULTSThe positivity rates of Beclin1 expression in the HCC, cirrhotic liver, hepatitis liver and normal liver tissues were 78.00% (39/50), 26.67% (8/30), 53.33% (16/30), and 10.00% (1/10), respectively, showing significant differences between them (chi(2)=28.31, P<0.05). Beclin1 expression was significantly higher in HCC tissues than in the cirrhotic, hepatitis and normal liver tissues (chi(2)=20.39, 5.31, and 14.41, respectively, P<0.05), and hepatitis tissues showed significantly higher Beclin1 expression than hepatic cirrhosis tissues and normal hepatic tissues (chi(2)=4.44 and 4.12, respectively, P<0.05).

CONCLUSIONThe abnormal expression of Beclin1 is closely associated with the pathogenesis and development of primary hepatocellular carcinoma, and may play an important role in this process.

Adult ; Aged ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Beclin-1 ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Female ; Humans ; Immunohistochemistry ; Liver Neoplasms ; metabolism ; pathology ; Male ; Membrane Proteins ; genetics ; metabolism ; Middle Aged

OBJECTIVETo study whether CO-Q10 can protect liver injury caused by acute on chronic liver failure (ACLF) by autophagy.

METHODSRats were separated into three groups: control group, acute on chronic liver failure (ACLF) and intervenient group, liver tissues were observed by optical microscopy and electron microscopy. The levels of Beclin-1 expression were determined by real-time PCR. And Western Blot.

RESULTSAreas of necrosis detected in intervenient group were alleviated than in ACLF significantly. Most mitochondrias had been degradated in ACLF group while alive in intervenient group. Real-time PCR and Western Blot revealed level of beclin-1 in ACLF was lower than control and intervenient group.

CONCLUSIONIntervenient group may ameliorate rat liver injury by promoting autophagy.

Animals ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Autophagy ; Beclin-1 ; Humans ; Liver Failure, Acute ; genetics ; metabolism ; physiopathology ; Male ; Mitochondria, Liver ; metabolism ; Rats ; Rats, Sprague-Dawley ; Ubiquinone ; analogs & derivatives ; metabolism

OBJECTIVE: To investigate the effect of BECN1 expression on proliferation and invasion of human multiple myeloma (MM) cell line RPMI-8226. METHODS: RPMI-8226 cells were cultured in vitro, the recombinant plasmid pcDNA3.1-BECN1 was constructed and transfected into RPMI-8226 cells, then the cells were divided into three groups: control group, negative transfection group and BECN1 transfection group. The expression of BECN1 mRNA in cells of each group was detected by real-time fluorescent quantitative polymerase chain reaction (qRT-PCR); the effect of BECN1 overexpression on cell proliferation inhibition rate was detected by cell counting kit 8 (CCK-8); the effect of BECN1 overexpression on the colony formation rate was detected by plate cloning assay; the effect of BECN1 overexpression on cell invasion was detected by Transwell assay; the effects of BECN1 overexpression on the expression of cell proliferation, invasion and autophagy-related proteins were detected by Western blot. RESULTS: The expression of BECN1 mRNA in BECN1 transfection group was significantly higher than that in control group and negative transfection group (P<0.05); the inhibition rate of cell proliferation and the expression of autophagy-related proteins Beclin1, Atg5 and invasion-related protein E-cadherin in BECN1 transfection group were significantly higher than those in control group and negative transfection group; the colony formation rate, invasion number and the expression of proliferation-related proteins CyclinD1, β-catenin and invasion-related protein N-cadherin were significantly lower than those in control group and negative transfection group (P<0.05). CONCLUSION Overexpression of BECN1 can inhibit the proliferation and invasion of human MM cells RPMI-8226, which may be a potential research target of MM.
Beclin-1/metabolism* ; Cadherins/metabolism* ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Humans ; Multiple Myeloma/genetics* ; RNA, Messenger/metabolism* ; Sincalide/metabolism* ; beta Catenin/genetics*

OBJECTIVETo observe autophagy induced by starvation in non-small cell lung cancer A459 and 95D cells.

METHODSA549 and 95D cells in logarithmic growth in 1640 medium were cultured in Earle's balanced salt solution (EBSS) for 0, 1, 2, 3, 4 or 5 h. Autophagosome formation in the cell culture was observed by MDC fluorescent staining, and the expression of microtubule-associated protein 1 light chain 3 (LC3) and Beclin1 in the cells were detected using Western blotting.

RESULTSCompared with the control cells, the cells with prolonged starvation showed increased MDC-positive cells and autophagosome formation. The expression of Beclin-1 and the LC3-II/LC3-I ratio also increased as the starvation prolonged, reaching the peak levels at 3 h and 4 h, respectively.

CONCLUSIONAutophagy can be induced by starvation in A549 and 95D cells in correlation with the expression of autophagy-related proteins LC3 and Beclin-1. These cell models of nutritional deficiency-induced autophagy may allow for a better understanding of the role of autophagy in the development of non-small cell lung cancer.

Apoptosis Regulatory Proteins ; metabolism ; Autophagy ; Beclin-1 ; Carcinoma, Non-Small-Cell Lung ; pathology ; Cell Line, Tumor ; Humans ; Membrane Proteins ; metabolism ; Microtubule-Associated Proteins ; metabolism