Effect of autophagy in SH-SY5Y cells injured by Aβ25-35

AIM: To explore whether the damage of neurons induced by amyloid β-protein (Aβ) is related to the regulation of autophagy and its mechanism based on Akt/mTOR pathway. METHODS: SH-SY5Y cells were incubated with Aβ_25-35 (5 μmol/L, 10 μmol/L, 15 μmol/L, 20 μmol/L and 25 μmol/L) for 24 h, and the cell viability was measured by MTT assay. The protein levels of LC3-I, LC3-II, Akt, p-Akt, mTOR and p-mTOR in the SH-SY5Y cells were determined by Western blot. After the SH-5Y5Y cells were incubated with autophagy inducer rapamycin (Rapa) or autophagy inhibitor 3-methyladenine (3-MA) combined with Aβ_25-35 for 24 h, the cell viability and related protein expression were detected by the same methods above mentioned. RESULTS: Each concentration of Aβ_25-35 damaged SH-SY5Y cells and decreased the viability of SH-SY5Y cells. Aβ_25-35 increased the expression of autophagy marker protein LC3-II, increased the level of LC3-II/LC3-I, and down-regulated the phosphorylation level of Akt and mTOR proteins (P<0.05). When combined with autophagy inducer Rapa, the cell viability was not significantly affected, the expression of LC3-II protein was increased, LC3-II/LC3-I was increased significantly, and p-mTOR/mTOR level was decreased (P<0.05). When combined with autophagy inhibitor 3-MA, the protein expression of LC3-II and the level of LC3-II/LC3-I showed a downward trend, while the level of p-Akt/Akt was decreased (P<0.05). CONCLUSION: Aβ_25-35 may induce SH-SY5Y cell autophagy and injury by down-regulating phosphorylation levels of Akt and mTOR proteins.     

Baicalein induces autophagy in breast cancer cells

AIM:To investigate the autophagy of breast cancer cells induced by baicalein and to explore its mechanism.METHODS:The effects of baicalein on the viability of MCF-7 cells and 4T1 cells were investigated by MTT assay,and the dosage of the drug was determined.The expression levels of microtubule-associated protein 1 light chain 3-Ⅱ(LC3-Ⅱ) and LC3-I in the MCF-7 cells and 4T1 cells treated with baicalein at doses of 25,50 and 100 μmol/L,or combined with autophagy inhibitor 3-methyladenine (3-MA) were determined by Western blot.In order to confirm the role of baicalein in autophagy,the effect of 3-MA on the apoptosis of both MCF-7 cells and 4T1 cells induced by baicalein was analyzed by flow cytometry.The protein levels of p-mTOR,mTOR,p-AKT and AKT were examined by Western blot and the role of AKT-mTOR pathway in the induction of autophagy in breast cancer induced by baicalein was determined by the combination of activators.RESULTS:Baicalein at 50 μmol/L and above doses significantly inhibited the viability of breast cancer cells in a dose-and time-dependent manner.The expression of LC3-Ⅱ/LC3-I in both MCF-7 cells and 4T1 cells was significantly enhanced after the action of baicalein,and the ratio of LC3-Ⅱ/LC3-I was significantly decreased after 3-MA addition.The results of flow cytometry showed that,compared with baicalein group,the combination of baicalein and 3-MA promoted the levels of necrosis and apoptosis.Moreover,the protein levels of p-mTOR and p-AKT were significantly decreased and were rescued by EGF,while their total protein levels were not changed.CONCLUSION:Baicalein induces autophagy through AKT-mTOR pathway both in MCF-7 cells and 4T1 cells.     

Shikonin induces apoptosis and autophagy of human cervical cancer HeLa cells by PI3K/Akt/mTOR signaling pathway

AIM:To observe the effects of shikonin on the apoptosis and autophagy of human cervical cancer HeLa cells, and to explore the possible role of PI3K/Akt/mTOR signaling pathway in these processes. METHODS:The HeLa cells were treated with shikonin, and the cell viability was detected by CCK-8 assay. The apoptosis was detected by Annexin V/PI double staining. The autophagosome was observed by transfection with GFP-LC3 into the HeLa cells. After the treatment with shikonin combined with autophagy inhibitor 3-methyladenine (3-MA) or apoptosis inhibitor Z-DEVD-FMK, the protein levels of autophagy-and apoptosis-related molecules microtuble-associated protein 1 light chain 3 (LC3) and cleaved caspase-3 in the HeLa cells were determined by Western blot. The protein levels of phosphorylated PI3K (p-PI3K), phosphorylated Akt (p-Akt) and phosphorylated mTOR (p-mTOR) were also determined by Western blot. RESULTS:Shikonin significantly inhibited the viability of HeLa cells (P<0.05). Compared with control group, shikonin significantly induced apoptosis of HeLa cells (P<0.05). The results of GFP-LC3 plasmid transfection analysis showed that green dot-like congregate autophagosomes appeared in the cytoplasm of the HeLa cells after shikonin treatment, while the autophagosomes were rarely observed in control group. Compared with shikonin group, LC3-Ⅱ/LC3-I was significantly decreased and cleaved caspase-3 was significantly increased in shikonin+3-MA group (P<0.05). Compared with shikonin group, LC3-Ⅱ/LC3-I was significantly increased and cleaved caspase-3 was significantly decreased in shikonin+Z-DEVD-FMK group (P<0.05). Compared with control group, shikonin significantly decreased the protein levels of p-PI3K, p-Akt and p-mTOR (P<0.05). CONCLUSION:The apoptosis and autophagy of the HeLa cells are induced by shikonin, these two processes are complementary. The mechanism may be related to inhibition of PI3K/Akt/mTOR signaling pathway.     

Tripchlorolide activates p-ERK and induces autophagy in lung cancer A549 cells

AIM: To investigate the effects of tripchlorolide (TP) on proliferation and autophagy of human lung cancer A549 cells, and explore its mechanism. METHODS: MTT assay was performed to analyze the effect of TP on the viability of human lung cancer A549 cells. The A549 cells were treated with TP, and their autophagy was observed under the fluorescence microscope through acridine orange staining. Green fluorescence spots were observed by fluorescence microscopy through GFP-LC3 plasmid transfection experiment. The levels of LC3 and p-ERK in the A549 cells after TP treatment were determined by Western blot. RESULTS: The viability of human lung cancer A549 cells was significantly inhibited by TP in a dose-time dependent manner (P<0.05). The number of the intracellular acidic follicles dyed with bright red fluorescence was significantly increased after TP treatment in A549 cells. The number of green dot-like congregate autophagosomes in cell cytoplasm was significantly increased after TP treatment in the A549 cells transfected with GFP-LC3 plasmid, while the normal treatment only induced a few cells with autophagosome formation. At the same time, we did not observe the dot-like congregate autophagosomes after TP treatment in the A549 cells transfected with GFP-control plasmid. Compared with control group, the expression of LC3-Ⅱ protein was up-regulated in A549 cells after TP treatment (P<0.01). Furthermore, treatment with TP in A549 cells for 48 h also led to a significant upregulation of phosphorylated form of ERK (P<0.01). In contrast, no significant change in the levels of total ERK protein was observed. Compared with 100 nmol/L TP group, TP+3-MA group down-regulated the protein levels of LC3-Ⅱ (P<0.01) and p-ERK (P<0.01) in the A549 cells. CONCLUSION: TP significantly inhibits the growth of A549 lung cancer cells and induces the autophagy, which may be correlated with upregulation of p-ERK protein.     

NVP-BEZ235 induces autophagy of polycystic kidney rat cholangiocytes

AIM: To explore the effect of dual PI3K/Akt/mTOR inhibitor NVP-BEZ235 on autophagy of polycystic kidney (PCK) rat cholangiocytes. METHODS: The protein levels of p-mTOR and p-Akt in the bile duct epithelial cells were examined by immunohistochemistry. The effect of NVP-BEZ235 on the viability of cholangiocytes was detected by WST-1 assay. The levels of PI3K/Akt/mTOR signaling pathway and autophagy-related proteins with NVP-BEZ235 treatment were determined by Western blot. The effects of LC3 and Beclin 1 silencing, and authophagy-specific inhibitor 3-methyladenine (3-MA) on the cell viability were analyzed by WST-1 assay. RESULTS: The protein levels of p-Akt and p-mTOR were highly increased in the bile duct epithelium of the PCK rats. NVP-BEZ235 significantly inhibited the viability of the cholangiocytes in a dose- and time-dependent manner (P<0.05). NVP-BEZ235 significantly reduced the levels of PI3K/Akt/mTOR signaling pathway-related proteins in the PCK rat cholangiocytes. NVP-BEZ235 upregulated the autophagy-specific proteins LC3 II and Beclin 1. The inhibitory effect of NVP-BEZ235 on the cell viability was weakened by treatment with 3-MA and knockdown of LC3 and Beclin 1 (P<0.01).CONCLUSION: The PI3K/Akt/mTOR inhibitor NVP-BEZ235 suppresses the viability of PCK rat cholangiocytes, and the mechanism is closely related with autophagy.     

Tetrandrine induces autophagy of ovarian cancer cells by inhibiting PI3K/Akt/mTOR signaling pathway

AIM To investigate the effect of tetrandrine on the autophagy of human ovarian serous cystadenocarcinoma SKOV3 cells, and to explore its molecular mechanism. METHODS The SKOV3 cells were treated with various concentrations of tetrandrine, and the cell viability was measured by MTT assay. The formation of autophagolysosomes was observed by acridine orange staining under fluorescence microscope. The protein levels of LC3, mTOR, p-mTOR, Akt and p-Akt in the SKOV3 cells were determined by Western blot. The viability of the SKOV3 cells treated with tetrandrine alone or combined with autophagy inhibitor 3-methyladenine (3-MA) were measured by MTT assay. RESULTS Tetrandrine significantly inhibited the viability of SKOV3 cells in a dose-dependent manner (P<0.01). The results of acridine orange fluorescence staining showed that the number of intracellular autophagolysosomes with bright red fluorescence in the SKOV3 cells was significantly increased after tetrandrine treatment, while the autophagolysosomes were rarely observed in control group. The protein levels of LC3-II and P62 in the SKOV3 cells were significantly increased after tetrandrine treatment (P<0.01). Furthermore, treatment with tetrandrine resulted in significant down-regulation of phosphorylated form of mTOR and AKT in the SKOV3 cells (P<0.01), while total mTOR and AKT protein levels were not changed. Finally, combination of tetrandrine and 3-MA significantly decreased the cell viability compared with using tetrandrine alone (P<0.01). CONCLUSION The autophagy of human ovarian cancer SKOV3 cells were induced by tetrandrine and the molecular mechanism may be related to inhibition of PI3K/Akt/mTOR signaling pathway．     

Runx2 promotes osteogenic differentiating C2C12 cells through inhibiting macroautophagy

AIM：To explore the relationship between macroautophagy and Runx2-induced osteogenic differentiation of C2C12 cells.METHODS：The cells of C2C12/Runx2Dox subline were used in the study, which expresses Runx2 in response to doxycycline (Dox). The cells were treated with Dox (10 mg/L) for 0 d, 1 d, 3 d, and 6 d, and the expression levels of LC3b, Beclin-1, p62, and LAMP-2 were detected at each time point using real-time qPCR. The LC3-I/LC3-II ratio was measured by Western blotting. The cells were treated with different concentrations of 3-methyladenine (3-MA) or rapamycin (Rap) for 14 days in the presence of Dox, and the alkaline phosphatase (ALP) activity was measured. The cells were treated with 3-MA (5 mmol/L) or Rap (10 μmol/L) for 1 d, 3 d, and 6 d in the presence of Dox, and the expression levels of ALP and osteocalcin (OC) were detected.RESULTS：The expression levels of LC3b and Beclin-1 were dramatically down-regulated during Runx2-induced osteogenic differentiation of C2C12 cells, and the levels of p62 and LAMP-2 were not changed during this process. The conversion of LC3-I into LC3-II was inhibited. The ALP activity was increased in 3-MA (5 mmol/L)-treated cells, but decreased in Rap (10 μmol/L)-treated cells. The expression levels of ALP and OC were up-regulated by 3-MA treatment and were down-regulated by Rap treatment.CONCLUSION：Runx2 induces osteogenic differentiation of C2C12 cells by inhibiting the formation of autophagosome through down-regulating LC3 and Beclin-1 as well as inhibiting the conversion of LC3-I to LC3-II.     

Pycnogenol suppresses TGF-β1-induced hepatic stellate cell activation via ERK-mediated autophagy inhibition

AIM: To explore the effect of Pycnogenol on transforming growth factor-β1 (TGF-β1)-induced hepatic stellate cell activation. METHODS: Cultured LX-2 cells were treated with 5 μg/L TGF-β1 and different concentrations (0, 10, 25 and 50 mg/L) of Pycnogenol. The viability of the LX-2 cells under the conditions with or without autophagy inhibitor 3-MA and ERK inhibitor PD98059 was determined by MTT assay. The protein levels of α-SMA, ColⅠ, TIMP-1, LC3-Ⅱ/Ⅰ, beclin 1, p-ERK1/2 and ERK1/2 were detected by Western blot. RESULTS: Compared with control group, 5 μg/L TGF-β1 treatment elevated the cell viability, and increased the protein levels of α-SMA, ColⅠ, TIMP-1, LC3-Ⅱ/Ⅰ, beclin 1, p-ERK1/2, and ERK1/2 in the LX-2 cells (P<0.05). However, these effects were reversed by Pycnogenol pretreatment in a dose-dependent manner and the inhibitory effect of 50 mg/L Pycnogenol was the most significant in the LX-2 cells (P<0.05). Furthermore, compared with TGF-β1 group, pretreatment with 50 mg/L Pycnogenol, 5 mmol/L 3-MA or 20 μmol/L PD98059 downregulated TGF-β1-induced cell viability and the protein levels of α-SMA and LC3-Ⅱ/Ⅰ in the LX-2 cells (P<0.05). CONCLUSION: Pycnogenol suppresses TGF-β1-induced hepatic stellate cell activation via p-ERK and autophagy inhibition.     

Oxidized low-density lipoprotein induces autophagy in macrophages via CD36-mediated oxidative stress

AIM: To investigate the effect of oxidized low-density lipoprotein (ox-LDL) on autophagy in macrophages and the underlying molecular mechanisms. METHODS: RAW264.7 macrophages were pretreated with 2 mg/L anti-CD36 monoclonal antibody (anti-CD36 mAb), 5 μmol/L diphenyleneiodonium (DPI), 3 mmol/L 3-methyladenine (3-MA) or 1 μmol/L rapamycin for 1 h and then treated with ox-LDL (100 mg/L) for 12 h. The viability of the cells was measured by MTT assay. The activities of lactic dehydrogenase (LDH) in the medium and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, superoxide dismutase (SOD) in the cells as well as the levels of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) were determined to characterize the membrane integrity and the oxidative stress, respectively. The protein levels of beclin-1 and microtubule-associated protein 1 light chain 3-II (LC3-II), 2 important molecular markers of autophagy, were examined by Western blotting. RESULTS: ox-LDL induced autophagy in RAW264.7 macrophages as assessed by upregulation of beclin-1 and LC3-II. Similar to 3-MA, an autophagy inhibitor, anti-CD36 mAb significantly inhibited the ox-LDL-induced upregulation of beclin-1 and LC3-II. Anti-CD36 mAb suppressed the ox-LDL-induced oxidative stress as revealed by decreased NADPH oxidase activation, ROS and MDA generation as well as increased SOD activity. Similar results were observed in the cells pretreated with DPI, a NADPH oxidase inhibitor. Moreover, DPI significantly inhibited the ox-LDL-induced upregulation of beclin-1 and LC3-II. Inaddition, the decrease in the cell viability and increase in LDH release induced by ox-LDL were promoted by 3-MA and blocked by rapamycin (an autophagy inducer). CONCLUSION: ox-LDL induces autophagy in RAW264.7 macrophages, which may be involved in CD36-mediated ox-LDL uptake and subsequent activation of oxidative stress, and moderate activation of autophagy may protect macrophages from ox-LDL-induced injury.     

Effect of berberine on endoplasmic reticulum stress-autophagy pathway in human ovarian cancer SKOV3 cells

AIM: To investigate the regulatory effect of berberine on the endoplasmic reticulum stress-auto-phagy pathway in human ovarian cancer SKOV3 cells. METHODS: Human ovarian cancer SKOV3 cells were cultured in vitro, and berberine at doses of 12.5, 25, 50, 100, 200 and 400 μmol/L were added. After exposure for 12 h, 24 h and 48 h, the viability of the SKOV3 cells was measured by MTT assay. The cells were divided into control group, berberine (50 μmol/L) group, berberine (100 μmol/L) group, and berberine (200 μmol/L) group. After treatment with berberine for 24 h, the effects of berberine on the morphological changes of SKOV3 cells were observed under inverted phase-contrast microscope. The protein expression of microtubule-associated protein 1 light chain 3 (LC3) and ubiquitin-binding protein p62 was observed by indirect immunofluorescence method under laser confocal microscope. The protein expression of beclin-1,LC3,p62, CCAAT/lenhancer binding protein homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) was determined by Western blot. RESULTS: Berberine at 12.5, 25, 50, 100, 200 and 400 μmol/L significantly decreased the viability of SKOV3 cells at 12 h, 24 h and 48 h, and the IC₅₀ values of 12 h, 24 h and 48 h were (764.7±0.3) μmol/L, (231.6±0.1) μmol/L and (96.2±0.1) μmol/L, respectively. Laser confocal microscopy showed that the LC3 and p62 proteins were scattered and the fluorescence intensity was increased, while the point-like aggregation was also observed. Berberine at 200 μmol/L obviously enhanced the co-localization of LC3 and p62 proteins. Compared with control group, the expression of endoplasmic reticulum stress-related proteins GRP78 and CHOP, and autophagy-related proteins beclin-1, LC3 and p62 in berberine (200 μmol/L) group was increased significantly (P<0.05). CONCLUSION: Berberine may promote endoplasmic reticulum stress in SKOV3 cells by regulating autophagy.     

Angiotensin II type 1 receptor autoantibodies induces INS-1 islet β-cell apoptosis by upregulation of autophagy

AIM: To explore whether the angiotensin Ⅱ type 1 receptor autoantibodies (AT1-AA) induces islet β-cell apoptosis and whether autophagy is involved in the process. METHODS: The INS-1 cells treated with AT1-AA at 10^-6 mol/L for 24 h, and then the apoptosis was analyzed by flow cytometry, Western blot and Hoechst 33258 staining. In addition, the expression of autophagy-related proteins such as LC3 and beclin 1 were determined by Western blot. The effects of AT1-AA on the apoptosis, autophagy and viability of INS-1 cells with or without 3-methyladenine (3-MA; a common autophagy inhibitor) or telmisartan (an angiotensin Ⅱ type 1 receptor blocker) pretreatment, were detected by flow cytometry, Western blot and CCK-8 assay. RESULTS: Treatment with AT1-AA at 10^-6 mol/L for 24 h significantly reduced the cell viability (P<0.05). Compared with the negative IgG control group, the apoptotic cells increased after incubation with AT1-AA for 12 h, 24 h and 36 h, respectively (P<0.05). Moreover, the protein levels of LC3 and beclin 1 also increased gradually with the prolongation of treatment time, and the elevation of apoptosis and autophagy were blocked by telmisartan. After pretreatment with 3-MA, the apoptotic rate of the cells was obviously decreased compared with the cells treated with AT1-AA alone. CONCLUSION: AT1-AA induces the apoptosis of INS-1 islet β cells by upregulating autophagy via the angiotensin Ⅱ type 1 receptor pathway.     

Effects of chloroquine on autophagy and collagen metabolism in activated hepatic stellate cells in vitro

AIM: To explore the effects of chloroquine (CQ) on collagen Ⅰand collagen Ⅲ expression in activated rat hepatic stellate cell line HSC-T6 and the possible mechanism.METHODS: Transforming growth factor-β1 (TGF-β1) was used to activate HSC-T6 cells and 3 doses of CQ was administered for 24 h. The cells were divided into 5 groups as follows:control group, TGF-β1 group, TGF-β1+CQ (15 μmol/L) group, TGF-β1+CQ (30 μmol/L) group and TGF-β1 + CQ (60 μmol/L) group. Western blot was used to determine the expression of LC3-Ⅱ/LC3-I, P62 and α-SMA in activated HSC-T6 cells. The expression of collagen I and collagen Ⅲ was detected by immunocytochemical staining, Western blot and RT-qPCR. Western blot and RT-qPCR were also used to detect the expression of matrix metalloproteinase-13 (MMP-13), tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 at mRNA and protein levels.RESULTS: The ratio of LC3-Ⅱ/LC3-Ⅰ and P62 expression were increased after CQ intervention. Moreover, they were significantly higher in the TGF-β1+CQ groups than those in TGF-β1 group (P<0.01). The expression of collagen I and collagen Ⅲ at mRNA and protein levels was significantly increased in all TGF-β1+CQ groups as compared with TGF-β1 group (P<0.01), and it was markedly increased among TGF-β1+CQ groups in a dose-dependent manner. The expression of MMP-13 at mRNA and protein levels was significantly lowered and that of TIMP-1 and TIMP-2 was significantly increased in TGF-β1+CQ groups as compared with TGF-β1 group (P<0.05).CONCLUSION: Inhibition of autophagy by CQ in activated HSC-T6 cells up-regulates the expression of collagen I and collagen Ⅲ in a dose-dependent way, probably due to reduction of MMP-13 and enhancement of TIMP-1 and TIMP-2 expression.     

Autophagy protects macrophages from oxidized low-density lipoprotein-induced apoptosis by inhibiting C/EBP homologous protein expression

AIM: To investigate the protective effect of autophagy on oxidized low density lipoprotein (ox-LDL)-induced macrophage apoptosis and the underlying molecular mechanisms. METHODS: The RAW264.7 macrophages were pretreated with 3 mmol/L 3-methyladenine (3-MA), 1 μmol/L rapamycin (Rap) or 4 mmol/L 4-phenylbutyric acid (PBA) respectively for 1 h and then treated with ox-LDL (100 mg/L) for 12 h. The cell viability and apoptosis were determined by MTT assay and flow cytometry with Annexin V-FITC/PI staining, respectively. The activities of lactate dehydrogenase (LDH) in the medium and caspase-3 in the cells were determined by detection kits. The protein levels of beclin-1 (a molecular marker of autophagy), glucose-regulated protein 78 (GRP78, an endoplasmic reticulum stress marker) and C/EBP homologous protein (CHOP, a key-signaling component of endoplasmic reticulum stress-induced apoptosis) were examined by Western blot. Microtubule-associated protein 1 light chain 3 (LC3, another molecular marker of autophagy) was observed under laser scanning confocal microscope.RESULTS: Treatment of the RAW264.7 macrophages with ox-LDL at 100 mg/L for 12 h resulted in significant decrease in cell viability, and dramatic elevation in LDH leakage, cell apoptosis and caspase-3 activity, which were promoted by 3-MA (an autophagy inhibitor) and inhibited by Rap (an autophagy inducer). ox-LDL induced autophagy in the macrophages as assessed by beclin-1 upregulation and frequent granulation of LC3, which were inhibited by 3-MA and promoted by Rap. Interestingly, 3-MA enhanced, while Rap blocked, the CHOP upregulation induced by ox-LDL. Moreover, PBA (endoplasmic reticulum stress inhibitor) significantly inhibited ox-LDL-induced GRP78 upregulation and autophagy as determined by the attenuation of beclin-1 upregulation and frequent granulation of LC3. CONCLUSION: Endoplasmic reticulum stress mediates ox-LDL-induced autophagy in macrophages, and moderates activation of autophagy may protect macrophages from ox-LDL-induced apoptosis by inhibiting CHOP expression.     

Ellagic acid attenuates hypoxic-ischemic brain injury by alleviating autophagy

AIM:To investigate whether ellagic acid (EA) attenuates hypoxic-ischemic encephalopathy (HIE) by down-regulating autophagy. METHODS:In vivo, Sprague-Dawley rats (n=17) were randomly divided into 3 groups:5 rats for sham group, 6 rats for HIE group and 6 rats for HIE+EA pretreatment group. The rats in HIE+EA pretreatment group were treated with EA (10 mg/kg, 10 mL/kg, suspended in corn oil, ig). After 24 h of operation, the rats from each group were sacrificed and their brains were collected. TTC staining and HE staining were used to define the infarct areas and brain structure. The autophagy-related proteins beclin-1, P62, LC3-Ⅱ/-I and Atg5 in the cortex in each group were compared by Western blot. In vitro, PC12 cells were divided into 3 groups:control group, CoCl₂ group and CoCl₂+EA pretreatment group. CoCl₂ at 800 μmol/L was added to the PC12 cells to induce an anoxic environment. The PC12 cells were pretreated with EA at 8 μmol/L and the cell viability was measured by CCK-8 assay. The production of reactive oxidative species (ROS) in the cells was detected by flow cytometry with DCFH-DA staining. MDC staining and TMRE staining were applied to reflect the extent of autophagy and the state of apoptosis, respectively. The autophagy-related proteins in PC12 cells were also investigated. RESULTS:In HIE group, 7-day-old rats were given the operations and the their large infarct areas in the hemisphere were observed by TTC staining. HE staining displayed the injured hemispheres which contained few neurons, and exhibited edema status and serious structural damage. EA pretreatment decreased the infarct area and alleviated the damage to hemisphere with more visible neurons, compared with HIE group. Compared with sham group, the levels of autophagy-related proteins Atg5, beclin-1 and LC3-Ⅱ/-I in the cortex were increased (P<0.01), and P62 protein expression was decreased (P<0.01) in HIE group. Compared with HIE group, the protein expression of Atg5, beclin-1 and LC3-Ⅱ/-I was decreased (P<0.01) and P62 protein expression was increased in HIE+EA pretreatment group (P<0.01). In vitro, compared with CoCl₂ group, the PC12 cells in CoCl₂+EA pretreatment group showed a lower ROS level. Moreover, the cells in CoCl₂+EA pretreatment group exhibited higher mitochondrial membrane potential than that in CoCl₂ group. MDC staining in CoCl₂ group showed high value of fluorescence and increased number of autophagosomes. EA pretreatment reduced the number of autophagosomes and the extent of autophagy to protect PC12 cells. Furthermore, the protein levels of Atg5, beclin-1 and LC3-Ⅱ/-I in CoCl₂ group were higher (P<0.01), and the protein expression of P62 was lower (P<0.01) than those in control group. In CoCl₂+EA pretreatment group, the protein levels of Atg5, beclin-1 and LC3-Ⅱ/-I were decreased (P<0.01) and the protein expression of P62 was increased as compared with CoCl₂ group (P<0.01). CONCLUSION:EA pretreatment attenuates autophagy to protect the neurons against HIE injury.     

Role of autophagy in inhibition of human lung cancer PC9 cell proliferation by ursolic acid

AIM: To investigate the role of autophagy in inhibition of human lung cancer PC9 cell proliferation by ursolic acid (UA) as well as the underlying mechanism. METHODS: MTT assay and Trypan blue exclusion test were performed to analyze the effect of UA on the proliferation of PC9 cells. The PC9 cells were treated with UA, and autophagy was observed under fluorescence microscope through acridine orange staining. The expression of autophagy-associated proteins LC3 and ATG5 in the PC9 cells were detected by Western blot. The effect of UA, 3-methyladenine (3-MA) 3-MA or their combination on the cell viability was measured by MTT assay. RESULTS: The viability of PC9 cells was significantly inhibited by UA (P<0.05 or P<0.01). The number of bright red fluorescence positive cells was significantly increased after treatment with UA. The protein expression of LC3-Ⅱ and ATG5 was significantly up-regulated compared with control group (P<0.01). Furthermore, combination of UA and 3-MA resulted in a substantial decrease in cell viability compared with using UA alone (P<0.01). CONCLUSION: UA inhibits the proliferation and induces the autophagy of the PC9 cells, in which autophagy plays a protective role. The inhibition of autophagy significantly promotes the death of the PC9 cells induced by UA.     

Effect of autophagy on necroptosis of renal tubular epithelial cells in subtotal nephrectomy rats

AIM: To explore whether autophagy is involved in the excessive death of renal tubular epithelial cells in subtotal nephrectomy(SNx) rats and the relationship between autophagy and necroptosis in the kidney of SNx rats. METHODS: Male Sprague-Dawley rats were randomly assigned to control group(n=6) and SNx group(n=42). The rats in SNx group were subjected to SNx. Sham surgery was performed in the rats in control group. The rats in SNx group were divided into subgroups at 0, 4, 8 and 12 weeks(n=6) and the other rats in SNx group were divided into SNx+vehicle group, SNx+necrostatin-1(Nec-1) group and SNx+3-methyladenine(3-MA) group. The expression of RIP1, RIP3, LC3 and beclin-1 at mRNA and protein levels was measured at 0, 4, 8 and 12 weeks by qPCR and immunohistochemistry. The effects of Nec-1 or 3-MA on the protein expression of LC3-I, LC3-II and beclin-1, and production of reactive oxygen species(ROS) in the rat kidney were determined by Western blot and DCFH-DA staining. The death of renal tubular epithelial cells in the SNx rats was observed by TUNEL staining and electron microscopy. Finally, the effects of Nec-1 and 3-MA on blood urea nitrogen(BUN), serum creatinine(SCr) and the pathological changes of the renal tissues were analyzed. RESULTS: The highest mRNA and protein levels of RIP1, RIP3, LC3 and beclin-1 appeared at the 8th week after SNx(P<0.01). Compared with the rats in SNx+vehicle group, the protein over-expression of LC3-II/I and beclin-1, renal tubular epithelial cells with typical morphological features of necroptotic cell death and TUNEL-positive renal tubular cells were decreased in the SNx rats treated with Nec-1 and 3-MA(P<0.01), but 3-MA did not reduce the increased concentration of ROS. In addition, treatment with Nec-1 and 3-MA obviously reduced BUN, SCr(P<0.05), glomerulosclerosis index and tubulointerstitial injury score(P<0.01). CONCLUSION: Autophagy participates in the excessive death of renal tubular epithelial cells in SNx rats. Inhibition of autograph prevents necroptotic cell death of renal tubular cells, and alleviates chronic renal injury in SNx rats.     

Anticancer function of Shp2 in lung adenocarcinoma A549 cells and rela-ted molecular mechanisms

AIM: To explore the anticancer function of Shp2 in lung adenocarcinoma A549 cells and the related molecular mechanisms. METHODS: The viability and proliferation of A549 cells treated with Shp2 specific inhibitor Phps-1 or cisplatin (DDP) were measured by CCK-8 assay and EdU assay. Annexin V-FITC/PI double staining was applied to detect apoptotic rate of A549 cells with different interventions. The protein levels of caspase-3-17p, Bcl-2, Bax, p-STAT3/STAT3 and p-ERK/ERK were determined by Western blot. RESULTS: Compared with control group, Phps-1 at the concentration of 20 μmol/L significantly increased the viability of A549 cells after 24 h of treatment (P<0.05). Meanwhile, the proliferation rate of A549 cells in Phps-1 20 μmol/L group was significant increased compared with control group (P<0.05). The apoptotic rate of A549 cells in DDP treatment group decreased from 13.01%±2.62% to 3.67%±0.93% after adding Phps-1 (P<0.05). Phps-1 down-regulated the protein levels of caspase-3-17p, Bax and p-ERK, but up-regulated p-STAT3.CONCLUSION: Shp2 is a tumor suppressor in A549 cells, which may be associated with the activation of STAT3 signal pathway.     

AngiotensinⅡ activates autophagy pathway through elevating ROS level in vascular endothelial cells

AIM: To evaluate the effects of angiotensinⅡ (AngⅡ) on autophagy induction in vascular endothelial cells. METHODS: Human vascular endothelial EA.hy926 cells were used in the study. Intracellular reactive oxygen species (ROS) levels were detected by a microplate reader after the cells were treated with AngⅡ (10^-7 mol/L) or AngⅡ combined with antioxidant N-acetyl-L-cysteine (NAC,50 μmol/L) for 24 h. The protein levels of LC3-Ⅱ was detected by Western blotting after the cells were stimulated by different concentrations (10^-8, 10^-7, 10^-6 mol/L) of AngⅡ for 24 h or by AngⅡ (10^-7mol/L) for different time (0 h, 6 h, 12 h, 24 h, 36 h). The number of autophagosomes was evaluated by fluorescence microscopy after stained with acridine orange. Similarly, the protein level of LC3-Ⅱ and the number of autophagosomes were detected after treated with AngⅡ(10^-7mol/L), AngⅡ combined with autophagy inhibitor 3-methyladenine (3-MA) at concentration of 2 mmol/L or AngⅡ combined with NAC at concentration of 50 μmol/L. RESULTS: Intracellular ROS level and LC3-Ⅱprotein level were significantly increased (P<0.05) after the cells were treated with AngⅡ, accompanied by the significant increase in the number of autophagosomes. AngⅡ-induced autophagy (as showed both in LC3-Ⅱprotein level and autophagosomes) was dramatically down-regulated by the treatment with 3-MA or NAC in EA.hy926 cells (P<0.05). CONCLUSION: AngⅡ induces autophagy through elevating ROS levels in EA.hy926 cells.     

Effect of luteolin on TGF-β1-induced epithelial-mesenchymal transition in lung cancer A549 cells

AIM:To investigate the effects of luteolin on the invasion and epithelial-mesenchymal transition (EMT) induced by transforming growth factor-β1 (TGF-β1) in lung cancer A549 cells. METHODS:The effect of luteolin at 5, 10, 20, 40, 80 and 160 μmol/L on the viability of A549 cells was measured by MTT assay. The invasion ability was analyzed by Transwell method. The morphological changes of the A549 cells were observed under microscope.The protein expression of E-cadherin and vimentin in the A549 cells were determined by Western blot. RESULTS:The viability of the A549 cells was significantly inhibited by luteolin in a dose-time dependent manner (P<0.05). The IC₅₀ of luteolin for the A549 cells (24 h) was 68.79 μmol/L, while that (48 h) was 47.86 μmol/L. TGF-β1 induced morphological alteration of the A549 cells from epithelial to mesenchymal forms. Luteolin significantly inhibited TGF-β1-induced invasion of the A549 cells (P<0.01). The protein expression of E-cadherin was significantly down-regulated and the protein expression of vimentin was significantly up-regulated in the presence of TGF-β1 at 5 μg/L (P<0.01). However, luteolin reversed TGF-β1-induced EMT, up-regulation of E-cadherin and down-regulation of vimentin (P<0.01). CONCLUSION:Lu-teolin reverses TGF-β1-induced EMT in the lung cancer A549 cells.     

Abietic acid alleviates AGEs-induced apoptosis and endoplasmic reticulum stress in cardiomyocytes via inducing autophagy

AIM: This study aims to explore the effect of abietic acid (AA) on advanced glycosylation end products (AGEs)-induced apoptosis and endoplasmic reticulum stress in H9c2 cardiomyocytes. METHODS: H9c2 cells were divided into 5 groups. The cells in control group were treated with saline for 24 h. The cells in AGEs treatment group were treated with AGEs (100 mg/L) for 24 h. The cells in AGEs+AA (10, 25 and 50 μmol/L) groups were simulta-neously treated with AGEs (100 mg/L) and AA (10, 25 and 50 μmol/L) for 24 h. The cell viability was measured by MTT assay. The protein levels of myoglobin (Mb), creatine kinase MB isoenzyme (CK-MB), cardiac troponin I (cTnI), C/EBP homologous protein (CHOP), cleaved caspase-12, GADD34, BiP, LC3, P62 and beclin 1 were determined by Western blot. The levels of lactate dehydrogenase (LDH) were measured by ELASA. The apoptosis was analyzed by flow cytometry. RESULTS: The low concentration (<50 μmol/L) of abietic acid had no obvious effect on the viability of H9c2 cells. The high concentration (>50 μmol/L) of abietic acid decreased the viability of H9c2 cells. The levels of Mb, CK-MB, cTnI and LDH in AGEs group were higher than those in control group (P<0.05). Compared with AGEs group, the levels of Mb, CK-MB, cTnI and LDH in AGEs+AA (10, 25 and 50 μmol/L) groups were obviously reduced (P<0.05). Abietic acid at concentrations of 10, 25 and 50 μmol/L inhibited AGEs-induced apoptosis, elevated the protein levels of CHOP and cleaved caspase-12, and attenuated expression of GADD34 and BiP (P<0.05). Moreover, abietic acid at concentrations of 10, 25 and 50 μmol/L suppressed AGEs-induced decreased ratio of LC3-Ⅱ/LC3-Ⅰ and expression of beclin 1, and enhanced the expression of P62 (P<0.05). 3-Methyladenine, an inhibitor of autophagy, reversed the effect of abietic acid on the protein levels of LC3, Mb, cleaved caspase-12 and BiP (P<0.05). CONCLUSION: Abietic acid alleviates AGEs-induced apoptosis and endoplasmic reticulum stress in H9c2 cardiomyocytes via inducing autophagy.