Angiotensin 1-7 attenuates angiotensin Ⅱ-induced human glomerular endothelial cell injury via Mas receptor

AIM:To investigate the effect of angiotensin 1-7 (Ang1-7) on the human glomerular endothelial cells (HGECs) injury induced by angiotensin Ⅱ (Ang Ⅱ) and its possible mechanism. METHODS:Cultured HGECs were divided into 6 groups randomly:control group, Ang Ⅱ group, Ang1-7 group, Ang Ⅱ +Ang1-7 group, Ang Ⅱ +Ang1-7+A779 (an inhibitor of Mas receptor) group and A779 group. The apoptotic rate and reactive oxygen species (ROS) of HGECs were analyzed by flow cytometry and photographed by fluorescence microscopy. The levels of lactate dehydrogenase (LDH), nitric oxide (NO), endothelin-1 (ET-1), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β), monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule-1 (ICAM-1) in the supernatant of cell cultures were measured. RESULTS:Compared with the control group, the apoptotic rate and the average fluorescence intensity of ROS were increased in the Ang Ⅱ group, IL-6, TNF-α, TGF-β, ICAM-1 and MCP-1 in cell supernatants were also increased in the Ang Ⅱ group (P<0.05). Compared with the Ang Ⅱ group, the apoptotic rate, ROS level, and the above inflammatory factors were decreased in Ang Ⅱ +Ang1-7 group (P<0.05). Compared with the Ang Ⅱ +Ang1-7 group, adding A779 increased the cell apoptotic rate, ROS production and the releases of the above inflammatory factors in cell supernatants (P<0.05). Compared with the Ang Ⅱ group, adding Ang1-7 inhibited the LDH leakage, ET-1 secretion and promoted the release of NO in a dose-dependent manner (P<0.05). CONCLUSION:Ang1-7 attenuates the HGECs injury induced by Ang Ⅱ by inhibiting the Mas receptor.     

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.     

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.     

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.     

Astragaloside IV attenuates apoptosis of H9c2 cardiomyocytes induced by angiotensin II

AIM: To investigate the protective effect of astragaloside IV (ASIV) on angiotensin II (Ang II)- induced apoptosis of H9c2 cardiomyocytes. METHODS: H9c2 cardiomyocytes were treated with different concentrations of Ang II and ASIV. The effects of Ang II and ASIV on the viability of H9c2 cells was measured by CCK-8 assay. The optimum concentration of Ang II was 1 μmol/L and the concentrations of ASIV were 25, 50 and 100 μmol/L. The H9c2 cells was divided into 6 groups:control group, ASIV group, Ang II group, Ang II+ASIV (25 μmol/L) group, Ang II+ASIV 50 (μmol/L) group and Ang II+ASIV (100 μmol/L) group. The morphological changes of the H9c2 cells were observed under inverted phase-contrast microscope. Apoptosis was detected by TUNEL assay. The generation of reactive oxygen species (ROS) was detected by DCFH-DA staining. The protein expression of Bax, Bcl-2, nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) was determined by Western blot. H9c2 cardiomyocytes were transfected with negative control shRNA (NC) or Nrf2-shRNA (shRNA), and the cells were divided into 8 groups:NC+control group, NC+AngⅡgroup, NC+ASIV group, NC+AngⅡ+ASIV group, shRNA+control group, shRNA+AngⅡgroup, shRNA+ASIV group and shRNA+AngⅡ+ASIV group. ROS level was detected by ROS detection kit. The protein expression of Nrf2 and HO-1 was determined by Western blot. RESULTS: Ang II decreased the viability of H9c2 cells in a concentration-dependent manner (P<0.05). ASIV reversed the effect of Ang II on the viability of H9c2 cells in a concentration-dependent manner (P<0.05). Compared with control group, the apoptotic rate, the level of ROS and the protein expression of Bax in Ang II group were increased significantly, while the protein expression of Bcl-2, Nrf2 and HO-1 was decreased significantly (P<0.05). Compared with Ang II group, ASIV reversed the increase in apoptotic rate of H9c2 cells induced by Ang II in a concentration-dependent manner, reduced ROS level, down-regulated the protein expression of Bax and up-regulated the protein expression of Bcl-2, Nrf2 and HO-1 (P<0.05). After shRNA transfection, the effects of ASIV decreasing ROS production induced by Ang II and up-regulating the expression of Nrf2 and HO-1 were eliminated. CONCLUSION: ASIV protects H9c2 cardiomyocytes from apoptosis induced by Ang II, which may be related to reducing ROS generation and mediating the Nrf2/HO-1 signaling pathway.     

Over-expression of ACE2 gene alleviates Ang Ⅱ-induced oxidative stress in neural cells

AIM:To investigate the effect of over-expression of angiotensin-converting enzyme 2 (ACE2) gene on angiotensin Ⅱ (Ang Ⅱ)-induced oxidative stress and NADPH oxidase (NOX) expression in mouse neuroblastoma Neuro-2A cells. METHODS:The recombinant lentivirus encoding ACE2 gene was constructed and transfected into the Neuro-2A cells at a multiplicity of infection (MOI) of 10 for 72 h. The transfection efficiency of ACE2 gene and protein expression of ACE2 were detected, and the Neuro-2A cells were identified by detection of a neural cell marker. The Neuro-2A cells were divided into 7 groups:control group, eGFP group, ACE2-eGFP group, Ang Ⅱ treatment group, Ang Ⅱ-eGFP group, Ang Ⅱ-ACE2-eGFP group and Ang Ⅱ-ACE2-eGFP-A779 group. The Ang(1-7) level was determined by ELISA. The level of reactive oxygen species (ROS) in the cells was measured with a method of DHE staining. The protein expression of MAS receptor and NOX subunits (NOX2, NOX4, p47^phox and p67^phox) was detected by Western blot. RESULTS:Ang Ⅱ signi-ficantly increased ROS levels (P<0.01) and up-regulated the protein expression of NOX2, NOX4, p47^phox and p67^phox (P<0.01), but down-regulated MAS protein expression (P<0.01). Over-expression of ACE2 inhibited Ang Ⅱ-induced increase in ROS, down-regulated the protein expression of NOX2, NOX4, p47^phox and p67^phox,and still increased the Ang(1-7) level (P<0.01) and MAS receptor expression (P<0.01). An antagonist of the MAS receptor, A779, blocked the down-regulating effect of ACE2 on NOX expression (P<0.05). CONCLUSION:ACE2 over-expression antagonizes Ang Ⅱ-induced oxidative stress via MAS receptor in the neural 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.     

Effect of remote ischemic postconditioning on autophagy of hippocampal neural cells after cardiopulmonary resuscitation in rats

AIM: To observe the effect of remote ischemic post-conditioning (RIPostC) on autophagy of hippocampal neural cells after cardiopulmonary resuscitation (CPR) in rats. METHODS: Male SD rats (n=45) were randomly divided into sham operation group (sham group), cardiac arrest (CA)/CPR group and RIPostC group, with 15 rats in each group. A CPR model of asphyxiated CA was established by clamping the tracheal tube. Neurological deficit scoring (NDS) was performed at different time points after return of spontaneous circulation (ROSC). The rats were sacrificed 24 h after ROSC and hippocampal tissues were removed. Western blot was used to detect autophagy markers LC3-Ⅱ/LC3-I and beclin-1 in the hippocampal tissues. The apoptosis was detected by TUNEL method. The formation of LC3 particles was observed by immunofluorescence. The ultrastructural changes of autophagosomes and mitochondria were observed under transmission electron microscope. RESULTS: Compared with sham group, the NDS scores of CA/CPR group were decreased, the protein expression of LC3-Ⅱ/LC3-I and beclin-1 was increased (P<0.05), and the apoptosis of the neural cells was increased (P<0.05). Compared with CA/CPR group, the NDS scores in RIPostC group was increased, the protein expression of LC3-Ⅱ/LC3-I and beclin-1 was decreased (P<0.05), and the neural cell apoptosis was decreased (P<0.05). The number of LC3 particles was decreased, intracellular autophagosome number was reduced, and the mitochondrial structure damage was alleviated. CONCLUSION: Remote ischemic post-conditioning improves neurological function in rats after CPR, which may be related to inhibition of excessive autophagy in hippocampus.     

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.     

Effects of rapamycin on hydrogen peroxide-induced vascular endothelial cell senescence

AIM:To investigate the effects of rapamycin (Rapa) on hydrogen peroxide (H₂O₂)-induced vascular endothelial cell senescence and to explore the underlying mechanisms. METHODS:The human umbilical vascular endothelial cells (HUVECs) were divided into 4 groups:control group, senescence group, Rapa+H₂O₂ group and 3-methyladenine (3-MA)+H₂O₂ group. MTT assay was performed to assess the cell viability. Senescence-associated β-ga-lactosidase (SA-β-Gal) staining was performed to measure the senescent cells in each group. The subcellular structures were observed under transmission electron microscope (TEM). The protein levels of phosphorylated Rb (p-Rb), Rb, p21, LC3-Ⅱ and beclin-1 were determined by Western blot. RESULTS:Compared with control group, the cell viability in H₂O₂ group was significantly decreased accompanied with higher rate of SA-β-Gal staining positive cells (P<0.05) and markedly damaged structure. Additionally, the protein levels of p-Rb and p21 in senescence group were increased markedly compared with control group (P<0.05). However, the cells pre-treated with Rapa prior to stimulation with H₂O₂ showed increased viability, decreased number of senescent cells and decreased protein levels of p-Rb and p21 as compared with the cells stimulated with H₂O₂ alone (P<0.05). Moreover, the TEM observation showed that the structure of the cells in Rapa+H₂O₂ group was roughly normal and the autophagosome was captured, and the expression levels of beclin-1 and LC3-Ⅱ were increased (P<0.05). Conversely, pre-treatment with autophagy inhibitor 3-MA resulted in opposite results. The cell viability was decreased significantly, more senescent cells were stained blue, higher protein levels of p-Rb and p21 were detected (P<0.05), poor subcellular structures were captured, and no beclin-1 and LC3-Ⅱ was detected. CONCLUSION:Rapa may retard the senescence of HUVECs induced by H₂O₂, and promoting autophagy may be the underlying mechanism.     

Effects of AZD8055 on autophagy and apoptosis in cholangiocarcinoma cells

AIM: To explore the effects of mammalian target of rapamycin (mTOR) double inhibitor AZD8055 on autophagy and apoptosis of human cholangiocarcinoma cell line HuCCT1. METHODS: The effect of AZD8055 on the viability of HuCCT1 cells was detected by MTT assay. Autophagosome was detected by acridine orange (AO) staining. After treated with AZD8055, the expression levels of apoptosis-related proteins Bcl-2, Bax and cleaved caspase-3 and auto-phagy marker proteins beclin 1, LC3 and p62 were determined by Western blot. Apoptotic rate was analyzed by flow cyto-metry with Annexin V-FITC/PI double staining. RESULTS: AZD8055 significantly inhibited the viability of HuCCT1 cells (P<0.05). AO staining showed that AZD8055 significantly increased orange granules in the cytoplasm. After treated with AZD8055, compared with the control group, the protein level of beclin 1 and the ratio of LC3-Ⅱ/LC3-I were enhanced, while p62 was attenuated (P<0.05). The protein expression level of pro-apoptotic regulator Bax was down-regulated and anti-apoptotic regulator Bcl-2 was increased. The protein level of cleaved caspase-3 was reduced (P<0.05). The results of flow cytometry showed that AZD8055 inhibited cell apoptosis. CONCLUSION: AZD8055 inhibits the viability of cholangiocarcinoma cells, and the mechanism is closely related with autophagy induced by AZD8055.     

Effects of MEG3 and adenosine on autophagy and proliferation of HepG2 cells

AIM:To study the effects of maternal expressed gene 3 (MEG3) and adenosine on the autophagy and proliferation of human hepatocellular carcinoma HepG2 cells, and to explore the possible mechanisms of autophagy and the effect on the proliferation of HepG2 cells induced by MEG3 and adenosine. METHODS:HepG2 cells were cultured according to the conventional cultural method, and divided into control group, MEG3 group (the cells were transfected with MEG3 lentivirus), adenosine group (the cells were treated with 1 mmol/L adenosine) and MEG3+adenosine group (the cells were treated with 1 mmol/L adenosine and MEG3 lentivirus). The protein expression of LC3-Ⅱ, LC3-Ⅰ and mammalian target of rapamycin (mTOR) was determined by Western blot. MDC staining was used to observe the number of autophagosomes. The cell viability was measured by CCK-8 assay, and the number of viable cells were counted by automated cell counter. RESULTS:Compared with control group, LC3-Ⅱ expression and the ratio of LC3-Ⅱ/LC3-Ⅰ were decreased, mTOR expression was increased (P<0.05), the viability of HepG2 cells was decreased, and the number of autophagosomes were reduced in MEG3 group and adenosine group. In MEG3+adenosine group, LC3-Ⅱ expression and the ratio of LC3-Ⅱ/LC3-Ⅰ were decreased significantly (P<0.01), mTOR expression was increased significantly (P<0.01), and the viability and autophagosomes of HepG2 cells were reduced markedly as compared with MEG3 group and adenosine group. After treated with MEG3 and adenosine for 24~72 h, the viable HepG2 cells reduced significantly in MEG3 group and adenosine group (P<0.01), especially in MEG3+adenosine group (P<0.01). CONCLUSION:MEG3 overexpression and low concentration of adenosine activate the mTOR pathway, and inhibit the autophagy and proli-feration of HepG2 cells. MEG3 enhances the effect of adenosine on HepG2 cells.     

Effects of resveratrol on viability,invasion and autophagy of osteosarcoma MG-63 cells by up-regulating miR-34a

AIM: To investigate the effects of resveratrol on the viability, invasion and autophagy of osteosarcoma MG-63 cells and the regulatory effect of microRNA-34a (miR-34a). METHODS: MG-63 cells were divided into 6 groups:control group and resveratrol treatment groups at doses of 10, 20, 40, 60 and 80 μmol/L. MTT assay, Transwell chamber method and Western blot were used to detect the effects of resveratrol on the viability, invasion ability and expression of autophagy-related proteins in the osteosarcoma cells. The effect of resveratrol at different concentrations on the expression of miR-34a in the osteosarcoma cells was detected by RT-qPCR. The effects of miR-34a mimic and miR-34a mimic negative control (miR-34a NC) transfection on the viability and invasion ability of osteosarcoma cells after treated with resveratrol at different concentrations were analyzed. The effects of miR-34a mimic transfection on autophagy-related proteins LC3-I, LC3-Ⅱ and beclin-1 were determined by Western blot. RESULTS: Compared with control group, resveratrol inhibited the viability and invasion ability of the MG-63 cells and promoted autophagy (P<0.05). Resveratrol up-regulated the expression of miR-34a in the MG-63 cells in a dose-dependent manner (P<0.05). In addition, the ratio of LC3-Ⅱ/LC3-I was increased, and beclin-1 was up-regulated (P<0.05). Co-treatment with miR-34a mimic and resveratrol increased inhibitory effects of resveratrol on the viability and invasion ability and invasion of the MG-63 cells and also promoted autophagy. CONCLUSION: Resveratrol inhibits the viability and invasion of osteosarcoma MG-63 cells and promotes auto-phagy by up-regulating miR-34a expression.     

Rapamycin reduces SH-SY5Y cell damage induced by oxygen-glucose deprivation

AIM: To observe the effect of rapamycin (Rapa) on human neuroblastoma SH-SY5Y cell injury induced by oxygen-glucose deprivation (OGD), and to explore the role of autophagy in this process. METHODS: The SH-SY5Y cells were randomly divided into 4 groups:normal control group:the cells were cultured without OGD treatment; Rapa group:the cells were pretreated with Rapa for 1 h; OGD group:the culture medium was replaced by glucose-free medium and the cells were transferred to a humidified incubation chamber flushed by a gas mixture of 1% O₂, 94% N₂ and 5% CO₂ for 12 h; Rapa+OGD group:the cultured cells were treated with Rapa for 1 h, and then were given the same treatments as those in OGD group. The cell viability was assessed by MTT assay. The degree of the cell damage was evaluated by determining the leakage of lactate dehydrogenase (LDH). The enzyme activity of caspase-3 was detected. TUNEL staining were used to detect the variation of cell apoptosis. The protein levels of apoptosis-related proteins Bax and Bcl-2, autophagy-related protein beclin-1 and autophagy marker protein LC3B were determined by Western blot. RESULTS: Compared with OGD group, the viability of the SH-SY5Y cells was significantly increased, and the activity of caspase-3 was significantly reduced in Rapa+OGD group (P<0.05). The SH-SY5Y cell injury was apparent after OGD with a great increase in the apoptotic rate (P<0.05). Compared with OGD group, the apoptotic rate significantly decreased in Rapa+OGD group (P<0.05). Compared with control group, the protein level of Bcl-2 was significantly decreased (P<0.05) and the protein level of Bax was significantly increased in OGD group. Compared with OGD group, the levels of Bcl-2, beclin-1 and LC3B-Ⅱ were significantly increased and the protein level of Bax was significantly increased in Rapa+OGD group (P<0.05). CONCLUSION: Rapamycin has a protective effect on in vitro cultured SH-SY5Y cells injured by OGD. The mechanism may be related to the promotion of autophagy.     

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.     

Lithium chloride promotes neuronal differentiation of rat bone marrow mesenchymal stem cells by modulating autophagy

AIM: To study the influence of lithium chloride (LiCl) on the neuronal differentiation of rat bone marrow mesenchymal stem cells (MSCs), and to explore whether autophagy was involved in this process. METHODS: MSCs were isolated and cultured in vitro. The cells were divided into LiCl group and control group. MSCs were treated with β-mercaptoethanol as an inducer for triggering the cells to differentiate into neurons. The expression of neuronal markers-neuron specific enolase (NSE) and microtubule-associated protein-2 (MAP-2), and autophagic marker-microtubule-associated protein 1 light chain 3 (LC3) were measured by immunofluorescence method and Western blot. An autophagy activator rapamycin and autophagy inhibitor 3-methyladenine (3-MA) were applied to modulate the autophagy in the LiCl treated-cells. The protein expression of NSE and MAP-2 were determined by Western blot. RESULTS: After induction, the expression of NSE and MAP-2 were increased. The percentage of NSE-and MAP-2-positive cells and the expression of NSE and MAP-2 in the LiCl group were greater than those in control group (P<0.05). After induction, the number of LC3-positive dots and the expression of LC3-Ⅱ in LiCl group were greater than those in control group (P<0.05). The expression of NSE and MAP-2 increased when the autophagy was modulated by rapamycin in LiCl treated-cells, and on the contrary, the expression of NSE and MAP-2 were inhibited as autophagy was modulated by 3-MA. CONCLUSION: Lithium chloride may promote the neuronal differentiation of rat bone marrow mesenchymal stem cells by modulating autophagy.     

Low-intensity ultrasound combined with microbubbles activates auto-phagic death of thyroid cancer cells by promoting ROS production

AIM: To investigate the effect of low-intensity ultrasound combined with microbubble contrast agent on autophagic death of thyroid cancer cells, and to analyze the mechanism of autophagy activation and its effect on cell viability. METHODS: Human thyroid cancer cell line TPC1 was treated with low-intensity ultrasound at 20 kHz frequency and 80 mW intensity combined with microbubbles. The cell death and viability were analyzed by Live/Dead assay and CCK-8 assay 60, 120 and 240 s after the treatment. The protein levels of microtubule-associated protein 1 light chain 3-Ⅱ (LC3-Ⅱ), autophagy-related protein 5 (ATG5) and SQSTM1/P62 were determined by Western blot. The number of intracellular autophagosomes was measured by the methods of monodansylcadaverine (MDC) staining, green fluorescent protein (GFP)-LC3 transfection and transmission electron microscopy. The level of reactive oxygen species (ROS) was mea-sured and the effect of ROS on autophagy activation was evaluated by N-acetyl-L-cysteine (NAC) treatment. The effect of ATG5 siRNA transfection on autophagy was analyzed for determining the role of autophagic death. RESULTS: Low-intensity ultrasound combined with microbubbles significantly promoted TPC1 cell death and inhibited TPC1 cell viability (P<0.05) in a time-dependent manner. Compared with low-intensity ultrasound group and microbubble group, ultrasound combined with microbubbles significantly increased the protein levels of LC3-Ⅱ and ATG5, but inhibited the protein level of P62 (P<0.05). The results of MDC staining, GFP-LC3 transfection and transmission electron microscopy showed that ultrasound combined with microbubbles significantly increased the number of autophagosomes in the TPC1 cells. Compared with low-intensity ultrasound group and microbubble group, ultrasound combined with microbubbles increased the level of ROS, while NAC significantly reduced the protein level of LC3-Ⅱ (P<0.05). Thansfection with ATG5 siRNA inhibited the autophagy, significantly decreased the percentage of cell death and increased cell viability (P<0.05). CONCLUSION: Low-intensity ultrasound combined with microbubbles promotes the autophagic cell death by increasing the level of ROS in thyroid cancer cells, leading to death of thyroid cancer cells.     

Effect of cytarabine on curcumin-induced autophagy in human acute myeloid leukemia cells

AIM:To observe whether autophagy occurs in curcumin-induced human acute myeloid leukemia KG1a cells in the presence of chemotherapeutic drug cytarabine and the possible mechanism. METHODS:KG1a cells were cultured in vitro. The ultrastructural changes of the cells were observed under transmission electron microscope. Autophagy was detected by acridine orange staining. The cell viability was measured by MTT assay. The cell cycle distribution was analyzed by flow cytometry. The expression of autophagy-related molecules beclin-1 and LC3 at mRNA and protein le-vels was determined by RT-qPCR and Western blot. RESULTS:Curcumin dose-dependently inhibited the viability of KG1a cells (P<0.05). The growth inhibition rate in combination group was significantly higher than that in single reagent group and control group (P<0.01). Electron microscopical observation showed that curcumin induced the occurrence of autophagosomes, and cytarabine increased curcumin-induced autophagosomes. Acridine orange staining showed that the combined treatment with cytarabine increased the autophagy induced by curcumin, and the number of autophagic acid vesicles and cells containing autophagic acid vesicles were increased. Curcumin blocked the cell cycle in the G₀/G₁ phase. The mRNA expression levels of beclin-1 and LC3 in combination group were significantly higher than those in single reagent group and control group(P<0.01). The results of Western blot showed that the protein expression of beclin-1 was significantly up-regulated in combination group (P<0.05), and the ratio of LC3-Ⅱ/LC3-I was higher than that in control group (P<0.01). CONCLUSION:Curcumin inhibits the viability of KG1a cells and induces autophagy. Cytarabine promotes autophagy, which is superior to curcumin alone. It may be related to the up-regulation of beclin-1 and LC3-Ⅱ by the two reagents.