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1.
Article in Chinese | WPRIM | ID: wpr-772049

ABSTRACT

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.


Subject(s)
Aldehyde Dehydrogenase , Aldehyde Dehydrogenase, Mitochondrial , Metabolism , Animals , Animals, Newborn , Autophagy , Beclin-1 , Physiology , Fibroblasts , Glucose , Microtubule-Associated Proteins , Mitochondrial Proteins , Rats , Rats, Sprague-Dawley
2.
Journal of Experimental Hematology ; (6): 1556-1560, 2019.
Article in Chinese | WPRIM | ID: wpr-775686

ABSTRACT

OBJECTIVE@#To investigate the effect of eukaryotic translation initiation factor 4E(eIF4E) on the autophagy of CD138 plasma cells in multiple myeloma(MM).@*METHODS@#Multiple myeloma CD138 plasma cells were treated with eIF4E inhibitor 4EGI, the changes of autophagy-related factors LC3-II and Beclin1 were detected by fluorescent quantitative PCR and Western blot, the changes of cell proliferation inhibition were detected by MTT assay, and cell apoptosis was detected by flow cytometry.@*RESULTS@#Quantitative fluorescence PCR showed that after treatment of myeloma cells with 4EGI, the expression levels of LC3-II and Beclin1 mRNA gradually increased with the enhancomer of 4EGI concentration and the prolongation of action time, and the differences were statistically significant (48 h: LC3-Ⅱ,r=0.942, Beclin1,r=0.952; 80 μg/ml: LC3-Ⅱ,r=0.966, Beclin1,r=0.998); Western blot showed that with the enhancement of 4EGI concentration, the expression of LC3-II and Beclin1 protein gradually increased(LC3-Ⅱ,r=0.923, Beclin1,r=0.977); CCK-8 showed that the inhibition rate of cells gradually increased (r=0.996); the apoptotic rate of 4EGI-treated groups (23.23±4.47, 7.59±1.67, 2.03±0.19) was significantly different from that of control group (0.03±0.04) (P<0.05).@*CONCLUSION@#The inhibition of eIF4E can activate the autophagy of CD138 plasma cells in multiple myeloma and induce the death of myeloma cells.


Subject(s)
Autophagy , Beclin-1 , Cell Line, Tumor , Eukaryotic Initiation Factor-4E , Humans , Multiple Myeloma
3.
Neuroscience Bulletin ; (6): 336-346, 2019.
Article in English | WPRIM | ID: wpr-775445

ABSTRACT

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.


Subject(s)
Animals , Autophagy , Physiology , Beclin-1 , Metabolism , Brain , Metabolism , Pathology , Brain Ischemia , Metabolism , Pathology , Therapeutics , 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 Uses , Random Allocation , Rats, Sprague-Dawley , Rats, Transgenic , Reperfusion Injury , Metabolism , Pathology , Therapeutics
4.
Article in Chinese | WPRIM | ID: wpr-774347

ABSTRACT

OBJECTIVE@#To explore the changes of autophagic activity after resistance of U266 cells to bortezomib (Bor) and its mechanisms.@*METHODS@#The proliferation inhibition rate, 50% inhibitory concentration (IC), drug-resistance coefficient, drug-resistance reversed multiple by 3-methyladenine (3-MA) in U266 and U266/Bor cells treated with Bor were detected and calculated by using MTT method, then the proliferation inhibition curve was drawed. The Western blot was used to detect the expression of LC3-I, IC3-II, p-mTOR, Beclin-1, ATG5 and ATG7 proteins.@*RESULTS@#The Bor showed the its proliferation inhibition effect on U266 cells and U266/Bor cells, IC of Bor on U266 and U266/Bor cells on 24 hours were 35.7 nmol/L and 526.5 nmol/L respectively; the drug-resistance coefficient was 14.7; the drug-resistance reversed multiple by 3-MA was 2.7. The expression of LC3-II, Beclin11, ATG5 and ATG7 in U266/Bor cells was higher than that in U266 cells; after the treatment with Bor for 24 h, the expression levels of LC3-II, Beclin-1, ATG5 and ATG7 in U266 cells all decreased, as compared with levels before treatment; while the expression levels of LC3-II, Beclin-1, ATG5 and ATG7 in U266/Bor cells were higher than those before treatment. There were no significant difference of p-mTOR expression among U266, U266+Bor, U266/Bor, U266/Bor+Bor cells.@*CONCLUSION@#The increase of autophagy closely relates with resistance of U266 cells to bortezomib, moreover with up-regulation of Beclin-1, ATG5 and ATG7 expression.


Subject(s)
Apoptosis , Autophagy , Beclin-1 , Bortezomib , Cell Line, Tumor , Cell Proliferation , Drug Resistance, Neoplasm , Humans
5.
Article in Chinese | WPRIM | ID: wpr-813239

ABSTRACT

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.


Subject(s)
Apoptosis , Apoptosis Regulatory Proteins , Autophagy , Beclin-1 , Bone Neoplasms , Cell Line, Tumor , HMGA2 Protein , Metabolism , Humans , MicroRNAs , Genetics , Osteosarcoma
6.
Article in Chinese | WPRIM | ID: wpr-776540

ABSTRACT

OBJECTIVE@#To investigate the effects of Notch signal on hypoxic induction factor (HIF-1α) and autophagy-associated genes Beclin1, LC3I, LC3II in oxygen-glucose deprivation (OGD) induced myocardial cell injury.@*METHODS@#The OGD model was established using hypoxic culture box and hypoglycemic DMEM medium. The cells were divided into normal control group, OGD group, OGD + NC siRNA group, OGD + Notch1 siRNA group and OGD + HIF-1α siRNA group. Western blot was used to detect the interference effects of HIF-1α siRNA and Notch1 siRNA. The effects of Notch1 siRNA and HIF-1α siRNA on the activity of myocardial cells in OGD model were detected by the CCK-8 assay. The effects of Notch1 siRNA and HIF-1α siRNA on autophage-associated genes Beclin1, LC3I and LC3II expression were detected by Western blot.@*RESULTS@#The results of Western blot showed that HIF-1α siRNA could effectively knock down the expression of HIF-1α in myocardial cells in OGD model, and Notch1 siRNA could effectively knock down the expression of Notch1 and HIF-1α in myocardial cells in OGD model. The result of CCK-8 assay showed that Notch1 siRNA and HIF-1α siRNA reduced the activity of myocardial cells in OGD model, and there was no statistical difference between the two groups. Western blot results showed that Notch1 siRNA and HIF-1α siRNA could reduce the expressions of the autophagy-associated genes Beclin1, LC3I and LC3II, and reduce the ratio of LC3II to LC3I at mRNA level.@*CONCLUSION@#Notch1 plays a role in myocardial protection by regulating the expression of HIF-1α to regulate the autophagy in OGD model cells.


Subject(s)
Autophagy , Beclin-1 , Metabolism , Cell Hypoxia , Cells, Cultured , Glucose , Humans , Hypoxia-Inducible Factor 1, alpha Subunit , Metabolism , Microtubule-Associated Proteins , Metabolism , Myocytes, Cardiac , Cell Biology , Pathology , Oxygen , Receptors, Notch , Metabolism , Signal Transduction
7.
Article in Chinese | WPRIM | ID: wpr-772680

ABSTRACT

OBJECTIVE@#To investigate the expression of autophagy-related protein Beclin-1 and microtubule-associated protein 2 light chain 3 (LC3Ⅱ) in periodontal ligament cells in orthodontic tooth pressure areas.@*METHODS@#Sixty male SD rats were randomly divided into a blank control group and nine experimental groups. In the experimental groups, 0.392 N orthodontic force was used to move the first right upper molars for 15 min, 30 min, 1 h, 2 h, 4 h, 12 h, 1 d, 3 d, or 7 d. The blank control group did not receive any treatment. The rats were euthanized. Changes in the morphology of the periodontal membrane in the pressure areas were observed through hematoxylin and eosin (HE) staining. The expression levels of Beclin-1 and LC3Ⅱ were detected by immunohistochemical staining, and tartrate-resistant acid phosphatase (TRAP) staining was performed for the counting of osteoclasts.@*RESULTS@#The HE stains showed that the hyalinization of the periodontal ligament appeared in the pressure areas after 1 day of exertion and was gradually aggravated. The immunohistochemical stains showed that the expression levels of Beclin-1 and LC3Ⅱ in the experimental groups gradually increased, peaked after 1 h, and then gradually decreased. The expression levels peaked again after 1 d, then decreased to baseline levels at 7 d of exertion. Beclin-1 and LC3Ⅱ were expressed in the osteoclasts. The TRAP stains indicated that the number of osteoclasts started to increase after 1 day.@*CONCLUSIONS@#Autophagy may participate in the process of periodontal ligament reconstruction in orthodontic tooth pressure areas by mediating the hyalinization of periodontal ligament and affecting the biological effects of osteoclasts.


Subject(s)
Animals , Autophagy , Beclin-1 , Metabolism , Male , Microtubule-Associated Proteins , Metabolism , Osteoclasts , Periodontal Ligament , Metabolism , Random Allocation , Rats , Rats, Sprague-Dawley , Tooth Movement Techniques
8.
Neuroscience Bulletin ; (6): 1037-1046, 2018.
Article in English | WPRIM | ID: wpr-775486

ABSTRACT

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.


Subject(s)
Autophagy , 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
9.
Article in Chinese | WPRIM | ID: wpr-773797

ABSTRACT

OBJECTIVE@#To study whether tricalcium phosphate(TCP) wear particles cause injuries of periprosthetic osteocytes in the mouse calvaria, and to explain its molecular mechanism.@*METHODS@#Thirty six-week(ICR)male mice were randomly divided into sham group, model (TCP) group and 3-methyladenine (3-MA) group. A murine calvarial model of osteolysis was established by 30 mg of TCP wear particles implantation over the periosteum around the middle suture of calvaria in mice. On the second postoperative day, the autophagy specific inhibitor 3-MA (1.0 mg/kg) was subcutaneously injected to the calvaria in the 3-MA-treated mice every other day. After 2 weeks, blood and the calvaria were obtained. Micro-CT was used to detect bone mineral density(BMD), bone volume fraction (BVF) and porosity number. HE staining and flow cytometry were performed to analyze the viability and apoptosis of periprosthetic osteocytes. The serum levels of dentin matrix protein 1(DMP-1) and sclerostin (SOST) were determined by ELISA. The proteins expressions of DMP-1, SOST, Beclin-1 and microtuble-associated protein 1 light chain 3 (LC-3) were detected by Western blot in the calvaria osteocytes.@*RESULTS@#Compared with the sham group, the mice in the TCP group showed that a significant decrease in the viability of periprosthetic osteocytes, but obvious increases in number of osteocytes death and osteocytes apoptosis (<0.05), and in serum level and protein expression of SOST; significant decreases in serum level and protein expression of DMP-1 (<0.05), and remarkable up-regulation of autophagy-related factors beclin-1 and the conversion of LC3-Ⅱ from LC3-I in the calvaria osteocytes. Compared with TCP group, the mice in the 3-MA group showed that injuries of calvaria osteocytes were obviously aggravated, and osteocytes apoptosis was significantly increased (<0.05).@*CONCLUSIONS@#TCP wear particles can cause injuries of periprosthetic osteocytes via activation of apoptosis and autophagy, which promotes osteolysis around the prosthesis osteolysis and joint aseptic loosening.


Subject(s)
Animals , Apoptosis , Beclin-1 , Metabolism , Bone Density , Calcium Phosphates , Extracellular Matrix Proteins , Metabolism , Glycoproteins , Metabolism , Male , Mice , Mice, Inbred ICR , Microtubule-Associated Proteins , Metabolism , Osteocytes , Pathology , Osteolysis , Prostheses and Implants , Skull
10.
Article in Chinese | WPRIM | ID: wpr-773760

ABSTRACT

OBJECTIVE@#To observe the effect of hypoxia on autophagy in Beclin-1-knockdown SH-SY5Y cells by constructing a stable transfected SH-SY5Y cell lines of silencing Beclin-1 gene.@*METHODS@#Beclin-1shRNA lentiviral vector and negative control lentiviral vector were constructed; the vector was transfected into SH-SY5Y cells; then the expression of Beclin-1 mRNA was detected by RT-PCR, the level of Beclin-1 protein was detected by Western blot. CCK-8 method was used to determine the effect of Beclin-1 knockdown on the viability of SH-SY5Y cells. Next, the blank control, negative control and transfected cells were cultured under 21% normoxia and 5% hypoxia conditions. The expression of LC3 protein in each group was detected by Western blot and the autophagic bodies were observed by electron microscopy.@*RESULTS@#Beclin-1 shRNA significantly inhibited the expression of Beclin-1 mRNA and protein in SH-SY5Y cells; after silencing Beclin 1 gene, the survival rate of Beclin-1 shRNA group cells was no different from that of negative control (NC) group. After 5% hypoxia treatment, compared with NC group, the ratio of LC3Ⅱ/LC3Ⅰand the number of autophagy bodies were all decreased in Beclin-1 shRNA group.@*CONCLUSIONS@#Beclin-1 knockdown SH-SY5Y cell lines and negative control cell lines were successfully established. Lentivirus-mediated Beclin-1 shRNA has no effect on the viability of SH-SY5Y cells, but can inhibit hypoxia-induced autophagy.


Subject(s)
Apoptosis , Autophagy , Beclin-1 , Cell Hypoxia , Cell Line, Tumor , Humans , RNA, Small Interfering
11.
Journal of Experimental Hematology ; (6): 1688-1694, 2018.
Article in Chinese | WPRIM | ID: wpr-773035

ABSTRACT

OBJECTIVE@#To investigate relationship of miRNA-132, miRNA-256, miRNA-143 and miRNA-145 level with antophagy and apoptosis of multiple mgeloma cells.@*METHODS@#Human myeloma cell line U266 and normal CD138 plasma cells were selected and used for study and detection, the 45 cases of MM were enrolled in MM group, and 40 normal persons were sellectod in control group. The expression of miRNA-132, miRNA-125b, miRNA-143 and miRNA-145 were measured by using qPCR, the expressions of autophagy-related protein (LC3-Ⅱ, LC3-Ⅰ, P62, beclin-1) and apoptosis-related molecules (cleaved-Caspase3, cleaved-Caspase7, BCL-2, BAX) were measured by using Western blot, respectively. The rate of apoptosis was measured by using flow cytometry. The correlation of miRNA expression level with clinical-related indexes including M protein, hemoglobin, β2-MG, lactate dehydrogenase, albumin, creatinine and serum calcium was analyzed.@*RESULTS@#Compared with normal plasma cells, the expression of miRNA-132 and miRNA-125b in myeloma cells increased significantly (P0.05). The expressions of miRNA-132, miRNA-125b, miRNA-143 and miRNA-145 were significantly different between DS and ISS staging group, also between the patients with abnormal and normal chromosome karyotype (P<0.05). The miRNA-125b and miRNA-143 significantly correlated with the levels of β2-MG, albumin and hemoglobin (P<0.05).@*CONCLUSION@#The expressions of miRNA-132, miRNA-125b, miRNA-143 and miRNA-145 in patients with multiple myeloma closely relate with the clinical characteristics. Both over-expression of miRNA-125b and down-expression of miRNA-143 inhibit the apoptosis of myeloma cells by up-regulation of autophagy.


Subject(s)
Apoptosis , Autophagy , Beclin-1 , Cell Line, Tumor , Humans , MicroRNAs , Multiple Myeloma
12.
Article in English | WPRIM | ID: wpr-772782

ABSTRACT

OBJECTIVE@#To investigate the effect of tea polyphenols on cardiac function in rats with diabetic cardiomyopathy, and the mechanism by which tea polyphenols regulate autophagy in diabetic cardiomyopathy.@*METHODS@#Sixty Sprague-Dawley (SD) rats were randomly divided into six groups: a normal control group (NC), an obesity group (OB), a diabetic cardiomyopathy group (DCM), a tea polyphenol group (TP), an obesity tea polyphenol treatment group (OB-TP), and a diabetic cardiomyopathy tea polyphenol treatment group (DCM-TP). After successful modeling, serum glucose, cholesterol, and triglyceride levels were determined; cardiac structure and function were inspected by ultrasonic cardiography; myocardial pathology was examined by staining with hematoxylin-eosin; transmission electron microscopy was used to observe the morphology and quantity of autophagosomes; and expression levels of autophagy-related proteins LC3-II, SQSTM1/p62, and Beclin-1 were determined by Western blotting.@*RESULTS@#Compared to the NC group, the OB group had normal blood glucose and a high level of blood lipids; both blood glucose and lipids were increased in the DCM group; ultrasonic cardiograms showed that the fraction shortening was reduced in the DCM group. However, these were improved significantly in the DCM-TP group. Hematoxylin-eosin staining showed disordered cardiomyocytes and hypertrophy in the DCM group; however, no differences were found among the remaining groups. Transmission electron microscopy revealed that the numbers of autophagosomes in the DCM and OB-TP groups were obviously increased compared to the NC and OB groups; the number of autophagosomes in the DCM-TP group was reduced. Western blotting showed that the expression of LC3-II/I and Beclin-1 increased obviously, whereas the expression of SQSTM1/p62 was decreased in the DCM and OB-TP groups (P<0.05).@*CONCLUSIONS@#Tea polyphenols had an effect on diabetic cardiomyopathy in rat cardiac function and may alter the levels of autophagy to improve glucose and lipid metabolism in diabetes.


Subject(s)
Animals , Autophagy , Beclin-1 , Blood Glucose , Body Weight , Diabetic Cardiomyopathies , Drug Therapy , Pathology , Lipids , Blood , Male , Myocardium , Pathology , Polyphenols , Pharmacology , Rats , Rats, Sprague-Dawley , Tea , Chemistry
13.
Chinese Acupuncture & Moxibustion ; (12): 1195-1200, 2018.
Article in Chinese | WPRIM | ID: wpr-777304

ABSTRACT

OBJECTIVE@#To compare the effects of electroacupuncture (EA) at different time during reperfusion on the expression of autophagy-related protein Bcl-2 and Beclin1 in myocardial tissue in rats with myocardial ischemia reperfusion injury (MIRI), and to explore the autophagy-related mechanism of EA on protecting MIRI.@*METHODS@#A total of 72 SD rats were randomly divided into a sham operation group, a model group, a RA group, a RB group, a RC group and a RD group, 12 rats in each group. Except the sham operation group, the rats in the remaining groups were treated with ligating the left anterior descending artery (LAD) for 30 minutes followed by reperfusion to establish the model of MIRI. The rats in the sham operation group were treated with crossing a line through the LAD. The rats in the model group did not receive treatment. The rats in the RA group, RB group, RC group and RD group were treated with EA at "Neiguan" (PC 6) for 20 min, starting at 0 h, 0.5 h, 1 h, 2 h after reperfusion. Evans Blue-TTC double-staining was employed to evaluate myocardial infarct size; the serum CK-MB was detected by ELISA and the expression of Bcl-2 and Beclin1 protein in myocardial tissue were detected by Western blot.@*RESULTS@#Compared with the model group, the percentage of myocardial infarct size in the RB group, RC group and RD group was decreased significantly (all 0.05); the increasing of Bcl-2 in the RB group was more significant than that in RC group (0.05).@*CONCLUSION@#EA at different time during reperfusion could reduce myocardial infarct size in rats with MIRI, and EA at 0.5 h after reperfusion has best efficacy; this protective effect may be achieved by increasing Bcl-2 expression and reducing Beclin1 expression to inhibit overautophagy during reperfusion.


Subject(s)
Animals , Beclin-1 , Electroacupuncture , Humans , Myocardial Ischemia , Myocardial Reperfusion Injury , Myocardium , Rats , Rats, Sprague-Dawley
14.
Article in Chinese | WPRIM | ID: wpr-297181

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the expression of autophagic gene and circadian gene in the neurons of neonatal rats after hypoxic-ischemic brain damage and the mechanism of nerve injury induced by hypoxia/ischemia.</p><p><b>METHODS</b>Twelve Sprague-Dawley (SD) rats were randomly divided into hypoxic-ischemic (HI) group and sham-operation group, with 6 rats in each group. Ligation of the right common carotid artery and hypoxic treatment were performed to establish a model of hypoxic-ischemic brain damage. Western blot was used to measure the expression of the circadian protein Clock in the cortex and hippocampus. The neurons of the rats were cultured in vitro and randomly divided into oxygen glucose deprivation (OGD) group and control group. The neurons in the OGD group were treated with DMEM medium without glucose or serum to simulate ischemic state, and hypoxic treatment was performed to establish an in vitro model of hypoxic-ischemic brain damage. Western blot was used to measure the expression of autophagy-related proteins Beclin1 and LC3 and Clock protein at different time points. The changes in the expression of Beclin1 and LC3 were measured after the expression of Clock protein in neurons was inhibited by small interfering RNA technique.</p><p><b>RESULTS</b>The expression of autophagy-related proteins Beclin1 and LC3Ⅱ in neurons cultured in vitro displayed a rhythmic fluctuation; after OGD treatment, the expression of Beclin1 and LC3Ⅱ gradually increased over the time of treatment and no longer had a rhythmic fluctuation. Compared with the sham-operation group, the HI group had a significant reduction in the expression of Clock protein in the cortex and hippocampus (P<0.05). After OGD treatment, the neurons cultured in vitro had a significant reduction in the expression of Clock protein (P<0.05). Compared with the negative control group, the Clock gene inhibition group had significant reductions in the expression of Beclin1 and LC3Ⅱ (P<0.05).</p><p><b>CONCLUSIONS</b>Hypoxia/ischemia induces the disorder in the expression rhythm of autophagy-related proteins Beclin1 and LC3, and the mechanism may be associated with the fact that the circadian protein Clock participates in the regulation of the expression of Beclin1 and LC3.</p>


Subject(s)
Animals , Animals, Newborn , Autophagy , Genetics , Beclin-1 , Genetics , Circadian Rhythm , Female , Hypoxia-Ischemia, Brain , Metabolism , Male , Microtubule-Associated Proteins , Genetics , Neurons , Metabolism , Rats , Rats, Sprague-Dawley
15.
Article in Chinese | WPRIM | ID: wpr-300818

ABSTRACT

Autophagy is fundamental to maintain cellular homeostasis. As one kind of the most well-studied selective autophagy, autophagy of mitochondria (mitophagy)is crucial for the clearance of damaged mitochondria. Mitophagy dysfunction has been proved to be closely associated with many human diseases. Nix is a key protein for mitophagy during the maturation of reticulocytes. However, the detailed molecular mechanisms underlying Nix-mediated mitophagy are not fully understood. This article summarizes three possible working models of Nix in mitophagy induction. Firstly, Nix can interplay with Parkin, another important protein for mitophagy, to initiate mitophagy. Secondly, Nix can serve as a receptor for autophagy machinery by interacting with Atg8 family through its LIR motif. Finally, as a BH3-only protein, Nix can compete with Beclin-1 to bind other members of Bcl-2 family resulting in increased free Beclin-1 in cytosol, which further promotes autophagy flux.


Subject(s)
Autophagy , Genetics , Physiology , Autophagy-Related Protein 8 Family , Physiology , Beclin-1 , Physiology , Membrane Proteins , Physiology , Mitochondria , Genetics , Physiology , Mitophagy , Genetics , Physiology , Protein Interaction Domains and Motifs , Proto-Oncogene Proteins , Physiology , Proto-Oncogene Proteins c-bcl-2 , Tumor Suppressor Proteins , Physiology , Ubiquitin-Protein Ligases , Physiology
16.
Article in Chinese | WPRIM | ID: wpr-264004

ABSTRACT

<p><b>OBJECTIVE</b>To explore the relationship of gentamicin-induced cochlear damage with autophagy-related protein LC3, beclin1, Na(+-)K(+-)2Cl(-) cotransporter (NKCC1) mRNA and endothelin-1 (ET-1), and investigate the protective mechanism of PPTA against gentamicin-induced cochlear damage.</p><p><b>METHODS</b>Sixty guinea pigs were randomly divided into control group (with saline and artificial perilymph injections), model group (with gentamicin and artificial perilymph injections), concurrent treatment group (with gentamicin and PPTA injections), model control group (with artificial perilymph injection 7 days after gentamicin injection) and delayed treatment group (with PPTA injection 7 days after gentamicin injection). Saline and gentamicin (160 mg/kg) were injected intraperitoneally, and artificial perilymph and PPTA were injected into the otocysts on a daily basis for 7 consecutive days. Hearing impairment of the guinea pigs was analyzed with ABR, and the protein expressions of beclin1 and LC3 in cochlear tissue were tested. The expression of NKCC1 mRNA was detected with RT-PCR, and the expression of ET-1 was detected immunohistochemically.</p><p><b>RESULTS</b>The ABR thresholds in the model group and model control group were similar (P>0.05) , but significantly higher than those in the other 3 groups (P<0.05); the threshold was significantly lower in concurrent treatment group than in delayed treatment group (P<0.05). Compared with those in the other 4 groups, the expressions of LC3 II, beclin1, and NKCC1 mRNA were significantly increased in the model group (P<0.05); and those in delayed treatment group were significantly lower than those in the model control group (P<0.05). The expressions of ET-1 in the Corti organ, striavascularis and spiral ganglion were significantly higher in the model group but significantly lower in the control group than those in the other 4 groups; ET-1 expression was significantly lower in delayed treatment group than in the model control group.</p><p><b>CONCLUSION</b>PPTA offers protection against gantamicin-induced cochlear damage in guinea pigs by inhibiting cell autophagy and suppressing of NKCC1 and ET-1 expressions. Early intervention with PPTA produces better therapeutic effect, suggesting that gantamicin causes irreversible injury of the auditory cells.</p>


Subject(s)
3,4-Methylenedioxyamphetamine , Pharmacology , Animals , Apoptosis Regulatory Proteins , Metabolism , Beclin-1 , Cochlea , Endothelin-1 , Metabolism , Gentamicins , Guinea Pigs , Hearing Loss , Microtubule-Associated Proteins , Metabolism , Solute Carrier Family 12, Member 2 , Metabolism
17.
Article in English | WPRIM | ID: wpr-287182

ABSTRACT

<p><b>OBJECTIVE</b>To observe the deregulation of autophagy in diabetic peripheral neuropathy (DPN) and investigate whether Jinmaitong ( JMT) alleviates DPN by inducing autophagy.</p><p><b>METHODS</b>DPN models were established by streptozotocin-induced diabetic rats and Schwann cells (SCs) cultured in high glucose medium. The pathological morphology was observed by the improved Bielschowsky's nerve fiber axonal staining and the Luxol fast blue-neutral red myelin staining. The ultrastructure was observed by the transmission electron microscopy. Beclin1 level was detected by immunohistochemistry and Western blot. The proliferation of cultured SCs was detected by methylthiazolyldiphenyl-tetrazolium bromide.</p><p><b>RESULTS</b>Diabetic peripheral nerve tissues demonstrated pathological morphology and reduced autophagic structure, accompanied with down-regulation of Beclin1. JMT apparently alleviated the pathological morphology change and increased the autophagy [in vivo, Beclin1 integral optical density (IOD) value of the control group 86.6±17.7, DM 43.9±8.8, JMT 73.3 ±17.8, P<0.01 or P<0.05, in vitro Beclin1 IOD value of the glucose group 0.47±0.25 vs the control group 0.88±0.29, P<0.05]. Consequently, inhibition of autophagy by 3-methyladenine resulted in a time- and concentration-dependent decrease of the proliferation of SCs (P<0.05, P<0.01).</p><p><b>CONCLUSIONS</b>Down-regulation of autophagy in SCs might contribute to the pathogenesis of DPN. JMT alleviates diabetic peripheral nerve injury at least in part by inducing autophagy.</p>


Subject(s)
Animals , Autophagy , Axons , Pathology , Beclin-1 , Metabolism , Cell Proliferation , Cells, Cultured , Diabetes Mellitus, Experimental , Drug Therapy , Pathology , Diabetic Neuropathies , Drug Therapy , Pathology , Down-Regulation , Drugs, Chinese Herbal , Pharmacology , Therapeutic Uses , Glucose , Pharmacology , Immunohistochemistry , Male , Rats, Wistar , Schwann Cells , Pathology , Sciatic Nerve , Pathology , Staining and Labeling
18.
Article in Chinese | WPRIM | ID: wpr-328311

ABSTRACT

<p><b>OBJECTIVE</b>To observe the effect of Xinfeng Capsule (XFC) on ankylosing spondylitis (AS) patients' symptoms and signs, serum immunoglobulin levels, peripheral blood lymphocyte autophagy protein, autophagy gene, and to explore its mechanism.</p><p><b>METHODS</b>Totally 59 AS patients were assigned to the treatment group (39 cases) and the control group (20 cases) according to random digit table. Patients in the treatment group received XFC, 0.5 g each pill, three pills each time, 3 times per day, while those in the control group received sulfasalazine (SASP), 0.25 g per tablet, 4 tablets each time, twice per day. Three months consisted of one therapeutic course. Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and Bath Ankylosing Spondylitis Functional Index (BASFI) were statistically calculated. Serum immunoglobulins (IgG1, IgG2, IgG3, IgG4, IgA , SIgA, and IgM) were detected using ELISA. Changes of Beclin1, LC3-II, phosphatidylinositol 3-kinase (PI3K), Akt, the mammalian target of rapamycin (mTOR) were detected using Western blot. Serum autophagy related genes such as Atg1, Atg5, Atg12, Atg13, and Atg17 were detected using the polymerase chain reaction (PCR). The correlation between immunoglobulin subtypes and autophagy gene in AS patients using Spearman correlation.</p><p><b>RESULTS</b>Compared with before treatment, BASDAI, IgG1, lgG3, and IgA decreased (P < 0.01); PI3K, Akt, and mTOR protein expressions decreased (P < 0.01); ATG1, ATG12, ATG13, and ATG17 mRNA expressions decreased, ATG5 mRNA expression increased (P < 0.01) in the treatment group. But BASDAI, IgG1, and IgA levels decreased (P < 0.05, P < 0.01); PI3K, Akt, and mTOR protein expressions decreased (P < 0.05); ATG1 and ATG13 mRNA expressions decreased (P < 0.05, P < 0.01) in the control group. Compared with the control group, BASDAI, IgG1, and IgA levels decreased (P < 0.05); PI3K, Akt, mTOR protein expressions decreased (P < 0.01); ATG12 and ATG17 mRNA expression decreased, ATG5 mRNA expression increased (P < 0.01) in the XFC group. Correlation analysis showed AS patients' IgG1, IgG2, IgG3, IgA, SIgA, IgM had negative correlation with ATG17; IgG4 and ATG17 were positively correlated (P < 0.05, P < 0.01).</p><p><b>CONCLUSION</b>XFC could elevate clinical efficacy of AS patients and enhance their autophagy, which might be achieved by acting on PI3K/Akt/mTOR signal, affecting autophagy gene and autophagy protein expression, taking part in the regulation of proliferation and differentiation of lymphocyte B, and strengthen humoral immunity.</p>


Subject(s)
Apoptosis Regulatory Proteins , Metabolism , Autophagy , Beclin-1 , Capsules , Drugs, Chinese Herbal , Therapeutic Uses , Humans , Immunoglobulin A , Blood , Immunoglobulin G , Blood , Immunoglobulin M , Blood , Lymphocytes , Membrane Proteins , Metabolism , Phosphatidylinositol 3-Kinases , Metabolism , Spondylitis, Ankylosing , Drug Therapy , Sulfasalazine , Therapeutic Uses , TOR Serine-Threonine Kinases , Metabolism
19.
Article in Chinese | WPRIM | ID: wpr-360060

ABSTRACT

<p><b>OBJECTIVE</b>To explore the autophagy of RPMI8226 cells induced by As(2)O(3) and its possible mechanisms.</p><p><b>METHODS</b>RPMI8226 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.</p><p><b>RESULTS</b>Different 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).</p><p><b>CONCLUSION</b>As(2)O(3) can induce autophagy of RPMI8226 cells, and the mechanism may be associated with the upregulation of Beclin-1.</p>


Subject(s)
Apoptosis Regulatory Proteins , Metabolism , Arsenicals , Pharmacology , Autophagy , Beclin-1 , Cell Line, Tumor , Cell Proliferation , Humans , Membrane Proteins , Metabolism , Oxides , Pharmacology , Up-Regulation
20.
Article in Chinese | WPRIM | ID: wpr-815081

ABSTRACT

OBJECTIVE@#To explore the effects of 3-methyladenine (3-MA, an autophagy inhibitor) on sensitivities of nasopharyngeal carcinoma cells to radiotherapy and chemotherapy and the underlying mechanisms.
@*METHODS@#Cell proliferation was examined by MTT and colony formation assay, while cell apoptosis was evaluated by annexin V/PI double staining and 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) staining. Mitochondrial membrane potential was measured by commercial kit (JC-1). The expression of endoplasmic reticulum stress (ERS)-related protein, glucose-regulated protein 78 (GRP78) and autophagy-related protein beclin1, microtubule-associated protein 1 light chain 3 (LC3) were examined by Western blot.
@*RESULTS@#Cisplatin (DDP), ionizing radiation (IR) or tunicamycin (TM) treatment obviously inhibited the proliferation of HONE-1 cells in a concentration-dependent and time-dependent manner. Compared with control group, pretreatment with 1 mmol/L of 3-MA significantly 
reduced cell viability and enhanced the apoptosis in the DDP (6.00 μmol/L), 4.00 Gy IR or TM (1.00 μmol/L) groups. There was no significant difference in the apoptosis between the DDP (5.8%) and 4Gy IR (6.7%) groups. Compared with the control group, protein levels of GRP78, beclin1 and lipid-conjugated membrane-bound form (LC3-II) were significantly increased after the treatment of DDP, 4.00 Gy IR or TM, which were inhibited by pretreatment of 3-MA.
@*CONCLUSION@#3-MA can sensitize HONE-1 cells to chemotherapy and radiotherapy, which is related to prevention of endoplasmic reticulum stress-induced autophagy in nasopharyngeal carcinoma cells.


Subject(s)
Adenine , Pharmacology , Apoptosis , Apoptosis Regulatory Proteins , Metabolism , Autophagy , Beclin-1 , Carcinoma , Cell Line, Tumor , Radiation Effects , Cell Proliferation , Cell Survival , Cisplatin , Pharmacology , Endoplasmic Reticulum Stress , Heat-Shock Proteins , Metabolism , Humans , Membrane Potential, Mitochondrial , Membrane Proteins , Metabolism , Microtubule-Associated Proteins , Metabolism , Nasopharyngeal Carcinoma , Nasopharyngeal Neoplasms , Pathology , Radiation, Ionizing , Radiation-Sensitizing Agents , Pharmacology , Tunicamycin , Pharmacology
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