Protective effect of autophagy on nucleus pulposus cells under starvation

AIM: To explore the possibility that the starvation environment induces autophagy of nucleus pulposus cells. METHODS: Primary rat nucleus pulposus cells was cultured and stained with toluidine blue, Alcian blue and immunocytochemistry for typeⅡ collagen. The cultured cells were divided into 4 groups: control group, 3-methyladenine (3-MA)+DMEM group, 3-MA+EBSS group and EBSS group. The cells were detected for autophay using monodansylcadaverine (MDC) staining, electron microscopy and Western blotting. At the same time, the inhibitory rate and apoptotic rate of the cells were detected by Cell Counting Kit-8(CCK-8) assay and TUNEL staining, respectively. RESULTS: Compared with control group, the autophagosomes were observed in nucleus pulposus cells under electron microscope and fluorescence microscope in EBSS group, and the 3-MA+EBSS treatment suppressed the formation of autophagosomes. The results of Western blotting analysis showed that the ratios of LC3-II/LC3-I and Beclin-1/β-actin in EBSS treatment group were higher than those in control group and 3-MA+EBSS treatment group. However, the apoptotic rate of nucleus pulposus cells and the inhibitory rate of cell viability were increased in 3-MA+EBSS treatment group. CONCLUSION: Autophagy of nucleus pulposus cells is induced by nutrient starvation, and 3-MA suppresses the response. Autophagy may have a protective effect on nucleus pulposus cells under the condition of starvation.     

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 glucocorticoid induced autophagy on senescence in chondrocytes

AIM: To explore the effect of glucocorticoid on autophagy and senescence in the chondrocytes. METHODS: The collagen II in the normal chondrocytes isolated from the SD rats was checked. After stimulation with glucocorticoid, LysoTracker Red staining, MDC staining and Western blot were used to detect the level of autophagy in the chondrocytes. The mTOR pathway related molecules were investigated by Western blot. Cell senescence was analyzed by SA-β-gal staining. RESULTS: A dose-dependent increase in the number of autophagic vacuoles was observed in the dexamethasone-treated chondrocytes, which was demonstrated by the LysoTracker Red and MDC staining. The expression of LC3-II and beclin-1 was increased by dexamethasone, especially in the cells treated with dexamethasone for 4 d. However, P62 expression was decreased. SA-β-gal staining showed that the percentage of cell senescence was increased by dexamethasone. Surprisingly, the cell senescence induced by dexamethasone was exacerbated by the autophagic inhibitor 3-MA. CONCLUSION: Autophagy induced by dexamethasone protects chondrocyte from senescence. The mTOR pathway may be involved in the autophagy activation.     

Autophagy plays a protective role in advanced glycation end product-induced apoptosis of vascular endothelial cells

AIM: To investigate the effect of advanced glycation end products (AGEs) on autophagy in human umbilical endothelial cells (HUVECs) and to identify the role of autophagy in advanced glycation end product-induced cell apoptosis. METHODS: HUVECs were cultured and treated with AGEs or bovine serum albumin. The protein expression was detected by Western blotting. Autophagosomes were observed under electron microscope. The cell apoptotic rate was determined by flow cytometry. The cell viability was quantified by MTT assay. RESULTS: After treated with AGEs, the level of autophagy-associated protein LC3-Ⅱ in HUVECs was up-regulated, and the number of autophagosomes was increased. Compared with control group, the apoptotic rate of HUVECs increased and the viability of HUVECs was decreased in AGEs treatment group. Furthermore, pretreating the cells with an autophagy inhibitor 3-methyladenine aggravated these effects. The levels of phospho-protein kinase B(Akt) and phospho-mammalian target of rapamycin(mTOR) in HUVECs were also decreased by treatment with AGEs. Pretreatment with Akt activator insulin-like growth factor 1 (IGF-1) increased Akt phosphorylation and suppressed the AGE-induced LC3-Ⅱ expression. CONCLUSION: AGEs induce autophagy in HUVECs through PI3K/Akt/mTOR signal pathway. Autophagy plays a protective role in AGE-induced apoptosis in HUVECs.     

Starvation induces autophagy of hypertrophic scar fibroblasts

［ABSTRACT］AIM: To investigate the starvation-induced autophagy of hypertrophic scar fibroblasts. METHODS: Primary human fibroblasts from hypertrophic scars were isolated and the fibroblasts in logarithmic growth phase were cultured with amino acid-free Earle's balanced salt solution (EBSS) instead of the DMEM medium. The cells were collected at the time points of 0 h, 1 h, 2 h and 3 h after EBSS culture. The expression of microtubule-associated protein 1 light chain 3 (LC-3) and autophagy-related protein Beclin-1 was detected by Western blotting and qRT-PCR. Autopagosomes in fibroblasts were observed by electron microscopy and monodansylcadaverine (MDC) staining. RESULTS: The expression of LC3 and Beclin-1 increased at 1 h after starvation, reached to the highest level at 2 h after starvation, and then began to decline, which were still higher than that in control group. Compared with control group, the autopagosomes were observed in the fibroblasts under fluorescence microscope and election microscope at 2 h after starvation. CONCLUSION: Autophagy in fibroblasts of hypertrophic scars can be induced by starvation, which may be related to the formation of hypertrophic scars.     

Role of autophagy inhibitor 3-methyladenine in the injury of U251 cells induced by H2O2

AIM: To investigate the role of autophagy inhibitor 3-methyladenine(3-MA) in the injury of U251 glioma cells induced by H₂O₂. METHODS: The following groups in this study were set up: control group, 10 mmol/L 3-MA group, 1 mmol/L H₂O₂ group and 1 mmol/L H₂O₂ +10 mmol/L 3-MA group. The viability of U251 cells in each group was detected by MTT assay. Autophagic vacuoles in the cells were observed by staining with MDC. The cells were stained with Hoechst 33342 to determine the chromatin condensation. Cell apoptotic ratio was measured by flow cytometry analysis. RESULTS: Compared with control group, no effect of 3-MA on the viability of U251 cells was observed. In H₂O₂ group, the cell viability decreased and cell apoptotic ratio increased.The autophagic vacuoles and nuclear chromatin condensation in the cells were also detected. Compared with H₂O₂ group, addition of 3-MA inhibited the increase in autophagic vacuoles but exacerbated the apoptosis. CONCLUSION: Autophagy inhibitor 3-MA inhibits autophagy partially, but exacerbates apoptosis in U251 cells, indicating that autophagy exerts protective effect in the process of injury in U251 cells induced by H₂O₂.     

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.     

Sublytic C5b-9 induces protective autophagy in cultured podocytes

AIM: In podocytes, autophagy occurs at a high basal level and dysregulated autophagy is associa-ted with a variety of podocytopathies. This paper is to investigate the role of autophagy in sublytic C5b-9-induced podocyte injury. METHODS: Sublytic complement C5b-9 stimulation was used as an in vitro model. Autophagosomes were confirmed using monodansylcadaverine (MDC) staining. Immunoblotting was used to measure the change of autophagy-related markers. Cellular morphological changes were observed by Wright-Giemsa staining. Immunofluorescence staining and confocal microscopy were used to detect the expression and distribution of nephrin. The cell viability was assessed by methylthiazol tetrazolium (MTT) assay. The cell apoptosis was assessed by Annexin V-fluorescein isothiocyanate/PI staining. RESULTS: For ensuring sublytic complement injury, the maximal amounts of anti-podocyte antiserum and 160×-diluted normal human serum were used without inducing cell lysis (defined as >5% LDH release). Sublytic C5b-9 promoted autophagy of podocytes in vitro. The proautophagic effect of sublytic C5b-9 manifested in the form of accumulated MDC-labeled vesicles and enhanced the expression of LC3-Ⅱ. Autophagy inhibitor 3-methyladenosine (3-MA) promoted sublytic C5b-9-induced podocyte morphological abnormalities. Compared with the sublytic C5b-9-injured podocytes, 3-MA exposure further decreased the expression of nephrin. 3-MA enhanced sublytic C5b-9-induced podocyte apoptosis. CONCLUSION: Sublytic C5b-9 attack induces autophagy, which may play a protective role against complement-mediated podocyte injury.     

Proctective effect of microRNA-30a on regulating beclin-1 expression in hypoxia-reoxygenated neonatal rat cardiomyocytes

AIM: To explore the potential mechanism of microRNA-30a (miR-30a) overexpression in neonatal rat cardiomyocytes during hypoxia/reoxygenation (H/R). METHODS: The miR-30a overexpression was induced in primary neonatal rat cardiomyocytes by lentivirus transfection. The cardiomyocytes were divided into 5 groups: normal group, H/R group, LV-GFP+H/R group, LV-GFP-miR-30a+H/R group and 3-methyladenine(3-MA)+H/R group. The expression level of miR-30a after lentivirus transfection and H/R was determined by real-time PCR, while the protein levels of LC3 and Beclin-1 after H/R and lentivirus transfection were detected by Western blotting. The cardiomyocyte death after H/R were measured by TUNEL and PI staining. RESULTS: Compared with LV-GFP group, significant down-regulation of Beclin-1 protein level was observed in cardiomyocytes with miR-30a overexpression, while the protein levels of Beclin-1 and LC3 in the cardiomyocytes with miR-30a overexpression were down-regulated after H/R, and apoptosis of these cells were significantly decreased after H/R. CONCLUSION: The protein level of Beclin-1 is down-regulated in cardiomyocytes with miR-30a overexpression. Inhibition of autophagy decreases the cardiomyocyte death after H/R.     

Autophagy is activated during glutamic acid-induced cortical neuron injury

AIM: To investigate whether autophagy is activated during glutamic acid-induced neuron injury and the possible neuroprotective effect of 3-methyl adenine(3-MA) (an autophagy inhibitor).METHODS: Glutamic acid or 3-MA was added to the medium of cultured cortical neurons. Cell viability was measured by MTT assay. The formation of autophagosome was observed under transmission electron microscope. The marker protein light chain 3(LC3) for autophagy was detected by immunofluorescence assay and visualized under laser confocal microscope.RESULTS: The cell viability declined during glutamic acid treatment and the autophagosomes were increased. LC3, the marker protein of autophagy, also significantly increased. The autophagy level was lowered by 3-MA, and cell viability was increased.CONCLUSION: The results suggest that autophagy is activated during glutamic acid treatment and inhibition of autophagy may have neuroprotective effect. The autophagy inhibitor 3-MA may be a potential neuroprotective agent.     

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.     

Nuciferine promotes autophagy and reduces macrophage foaming by inhibiting PI3K/Akt/mTOR signaling pathway

AIM To investigate the effect of nuciferine （NUF) on the formation of foam cells and its possible molecular mechanism. METHODS Human monocyte-macrophage cell line THP-1 was induced by oxidized low-density lipoprotein (Ox-LDL) to establish foam cell model, and simultaneously treated with NUF at 5, 10 or 20 μmol/L. Oil red O staining was used and total cholesterol content was measured to observe the effect of NUF on foam cell formation. Autophagy flow was detected by immunofluorescence, and autophagosomes were detected by transmission electron microscopy. The protein levels of microtubule-associated protein 1 light chain 3 (LC3), P62, phosphorylated protein kinase B (p-Akt) and phosphorylated mammalian target of rapamycin (p-mTOR) were determined by Western blot. 3-Methyladenine (3-MA), an autophagy inhibitor, was used to inhibit autophagy and to observe whether NUF inhibited foam cell formation by regulating autophagy. RESULTS Compared with control group, the intracellular lipid deposition and total cholesterol content in Ox-LDL group were increased. Compared with Ox-LDL group, the intracellular lipid deposition and total cholesterol content in NUF group were decreased, while autophagy flow and number of autophagosomes were increased. The inhibitory effect of NUF on cell foaming was weakened after 3-MA treatment. Moreover, NUF decreased the protein levels of p-mTOR and p-Akt. CONCLUSION Nuciferine may promote autophagy by inhibiting phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR signaling pathway, thus reducing intracellular lipid deposition and formation of foam cells.     

Doxycycline upregulates autophagy to modulate the levels of inflammatory cytokines in LPS-stimulated THP-1 cells

AIM:To analyze the effect of autophagy on inflammatory response regulated by doxycycline in lipopolysaccharide (LPS)-stimulated THP-1 cells and to investigate its molecular mechanism. METHODS:A human monocyte/macrophage cell line THP-1 was stimulated with LPS to establish an cell model of inflammatory response, and the cells were treated with doxycycline. The cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8), in cell culture supernatant were measured by ELISA for evaluating the inflammatory levels. For determining the level of autophagy and its effect on inflammatory cell signaling pathways, the protein levels of LC3B, nuclear factor κB (NF-κB) and phosphorylated mammalian target of rapamycin (p-mTOR) were determined by Western blot. 3-Methyladenine (3-MA), an autophagy inhibitor, and rapamycin, an autophagy inducer, were used to study the effect of autophagy on inflammatory response regulated by doxycycline in LPS-stimulated THP-1 cells. RESULTS:The levels of TNF-α and IL-8 were increased rapidly and peaked at 12 h in LPS-stimulated THP-1 cells (P<0.05). Doxycycline significantly inhibited LPS-induced cytokine production in the THP-1 cells. Doxycycline up-regulated LPS-induced autophagy in THP-1 cells and doxycycline itself was an autophagy inducer. The protein levels of p-mTOR was up-regulated by LPS and down-regulated by doxycycline, suggesting that doxycycline induced autophagy via mTOR-dependent pathway while LPS through mTOR-independent pathway. Further studies showed that the combination of LPS, rapamycin and doxycycline inhibited the protein levels of NF-κB, and rapamycin increased the inhibitory effect of doxycycline on cytokine releases. Conversely, 3-MA, the autophagy inhibitor, attenuated the inhibitory effect of doxycycline on NF-κB and cytokine production. CONCLUSION:Autophagy is involved in the process of doxycycline modulating LPS-induced inflammatory response in the THP-1 cells.     

Effects of astragaloside IV on autophagy in rats with cerebral ischemia/reperfusion injury

AIM: To investigate the effects of astragaloside IV (AS-IV) on autophagy in rats with cerebral ischemia/reperfusion (I/R) injury. METHODS: The focal cerebral ischemia/reperfusion of rat left middle cerebral artery occlusion (MCAO) was induced by suture method. Male SD rats (n=70) were randomly divided into sham operation group, I/R group, solvent control group, AS-IV group, AS-IV+autophagy inhibitor (3-methyladenine, 3-MA) group, 3-MA group and autophagy activator (rapamycin, Rapa) group. Except for sham operation group, the rats in other groups were subjected to ischemia for 2 h and reperfusion for 24 h. The rats with successful modeling were selected according to Zea Longa scoring criteria. The volume of cerebral infarction was measured by TTC staining. The morphological changes of nerve cells in the rats were observed with Nissl staining. The phenomenon of autophagy was observed under transmission electron microscope. The protein expression of beclin-1 and LC3-Ⅱ was determined by Western blot. RESULTS: No neurological deficit in sham operation group was observed, and the cerebral infarction was not found. Compared with sham operation group, obvious cerebral infarction was observed, the Nissl bodies were small in size and number and stained light, typical autophagosomes were observed, and the protein expression of beclin-1 and LC3-Ⅱ was increased in I/R group (P<0.05). Compared with I/R group, the volume of cerebral infarction was decreased obviously, neurological deficit restored significantly, and the number of autophagosomes and the protein expression of beclin-1 and LC3-Ⅱ were increased in AS-IV group and Rapa group (P<0.05). However, no significant difference between solvent control group and I/R group was observed (P>0.05). Compared with AS-IV group, the neurological deficit was serious, the volume of cerebral infarction and the number of autophagosomes were increased, while the expression of beclin-1 and LC3-Ⅱ was decreased in AS-IV+3-MA group and 3-MA group (P<0.05). CONCLUSION: Astragaloside IV may play an important role in atte-nuating cerebral ischemia/reperfusion injury by activating autophagy.     

Hypoxic preconditioning alleviates oxygen-glucose deprivation in PC12 cells:the involvement of autophagy

AIM: To examined the effects of hypoxic preconditioning(HPC) on oxygen-glucose deprivation(OGD)-induced PC12 cells, and to investigate its possible mechanisms of autophagy.METHODS: Cultured PC12 cells were randomly divided into control group, HPC group, 3-methyladenine(3-MA) group, HPC+OGD group, 3-MA+HPC+OGD group and OGD group. CCK-8 assay was used to detect the cell viability. The caspase-3 activity was also tested. TUNEL staining and flow cytometry were used to detect the cell apoptosis. The protein levels of apoptosis-related protein caspase-3 and autophagy-marked protein LC3-2 and beclin-1 were determined by Western blot.RESULTS: Compared with control group, the viability of PC12 cells was significantly reduced, and the activity of caspase-3 was significantly increased in OGD group. Compared with 3-MA+ HPC+OGD group and OGD group, the viability of PC12 cells was significantly increased, and the activity of caspase-3 was significantly reduced in HPC+OGD group(P<0.05). The PC12 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 HPC+OGD group(P<0.05). Compared with control group, the protein level of cleaved caspase-3 was significantly increased in OGD group(P<0.05). Compared with OGD group, the protein level of cleaved caspase-3 was significantly decreased, and the levels of LC3-2 and beclin-1 were significantly increased in HPC+OGD group(P<0.05).CONCLUSION: OGD decreases cell survival and induces apoptosis.Activation of cell autophagy may be the mechanism by which hypoxic preconditioning protects the PC12 cells from OGD induced injury.     

Crosstalk between autophagy and apoptosis induced by Rip2 and its mechanisms in human pancreatic cancer cells

AIM To investigate the crosstalk between autophagy and apoptosis caused by receptor-interacting protein 2 (Rip2) and its underling mechanisms in human pancreatic cancer cells. METHODS Plasmids (pEGFP-C2 and pEGFP-Rip2) were transfected into human pancreatic cancer Panc-1 cells by jetPRIME method. The Panc-1 cells transfected with pEGFP-Rip2 were treated with 3-methyladenine (3-MA), an autophagy inhibitor. The apoptotic rate was analyzed by flow cytometry. The levels of apoptosis-associated proteins were measured by Western blot. The activity of caspase-8, -9 and -3 was examined by colorimetric method. Moreover, the Panc-1 cells transfected with pEGFP-Rip2 were treated with Z-VAD-FMK, a broad inhibitor of caspases. Subsequently, the levels of autophagy- and PI3K/Akt/mTOR signaling pathway-related proteins were assessed by Western blot. The autophagosomes were observed under transmission electron microscope. RESULTS (1) The apoptotic rate in pEGFP-Rip2 group markedly increased as compared with control group and pEGFP-C2 group, while the apoptotic rate in pEGFP-Rip2+3-MA group was further elevated compared with pEGFP-Rip2 group (P<0.05). Meanwhile, the protein levels of Fas, Bax and cytoplasmic cytochrome c (Cyt-c) were significantly increased, and the protein expression of Bcl-2 was markedly reduced in pEGFP-Rip2+3-MA group as compared with pEGFP-Rip2 group (P<0.05). The activity of caspase-8, -9 and -3 in pEGFP-Rip2+3-MA group was higher than that in pEGFP-Rip2 group. (2) The protein expression of beclin-1 and LC3-Ⅱ was significantly increased and more accumulated autophagosomes were observed under transmission electron microscope in pEGFP-Rip2+Z-VAD-FMK group as compared with pEGFP-Rip2 group. Furthermore, the protein levels of p-mTOR and p-Akt in pEGFP-Rip2+Z-VAD-FMK group were markedly reduced compared with pEGFP-Rip2 group, while no significant difference of mTOR and Akt protein expression was found between the 2 groups. CONCLUSION Inhibition of autophagy promotes apoptosis induced by Rip2 in the pancreatic cancer cells. Its mechanism may be associated with the further activation of the intrinsic and extrinsic apoptotic pathways. Suppression of apoptosis accelerates autophagy induced by Rip2 in the pancreatic cancer cells, and the mechanism may be related to the further down-regulation of PI3K/Akt/mTOR signaling pathways. There is a mutual antagonistic effect between autophagy and apoptosis caused by Rip2 in pancreatic cancer cells.     

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.     

Hydrogen inhibits C/EBP homologous protein-mediated macrophage apoptosis by activating autophagy

AIM: To investigate the protective effect of hydrogen (H₂) on oxidized low-density lipoprotein (ox-LDL)-induced macrophage apoptosis and the underlying molecular mechanisms. METHODS: H₂-saturated medium was added to murine RAW264.7 macrophages and the cells were pretreated with 5 mmol/L 3-methyladenine (3-MA) and 3 μmol/L rapamycin (Rap) for 1 h, and then treated with ox-LDL (100 mg/L) for 24 h. The cell viability and apoptosis were determined by MTT assay and Annexin V-FITC/PI staining, respectively. The activity of lactate dehydrogenase (LDH) in medium was detected. The protein levels of beclin-1 (a molecular marker of autophagy) and C/EBP homologous protein (CHOP, a key signaling component of endoplasmic reticulum stress-associaed apoptosis pathway) were determined by Western blot. Microtubule-associated protein 1 light chain 3 (LC3, another molecular marker of autophagy) was observed under laser scanning confocal microscope. RESULTS: Hydrogen attenuated the reduction of cell viability, LDH leakage, apoptosis and CHOP upregulation induced by ox-LDL. Hydrogen promoted ox-LDL-induced autophagy in macrophages as assessed by beclin-1 upregulation, and LC3 granulation, and this promotion effect of hydrogen was inhibited by 3-MA (an autophagy inhibitor) and further enhanced by Rap (an autophagy inducer). Moreover, the inhibitory effect of hydrogen on ox-LDL-induced macrophage apoptosis, reduction of cell viability and CHOP upregulation were also blocked by 3-MA and enhanced by Rap. Similar results were obtained in human THP-1-derived macrophages, as assessed by the inhibition of ox-LDL-induced apoptosis and CHOP upregulation, and the promotion of beclin-1 expression by hydrogen. CONCLUSION: Hydrogen may protect macrophages from ox-LDL-induced apoptosis by inhibiting CHOP expression, and the upstream mechanism may partially involved in the activation of autophagy.     

Honokiol induces autophagy by inhibiting mTOR signaling pathway in lung cancer A549 cells

AIM:To investigate whether honokiol induces the autophagy of human lung cancer A549 cells and to explore its mechanism. METHODS:The A549 cells were cultured in vitro, and treated with honokiol at different concentrations (0, 10, 20, 40, 60 and 80 μmol/L) for 48 h. MTT assay was performed to analyze the effect of honokiol on the viability of the A549 cells. The formation of autophagosome was observed by staining with acridine orange under fluorescence microscope. The protein levels of autophagy-associated protein LC3, mTOR and p-mTOR in the A549 cells treated with honokiol, or combined with autophagy inhibitor 3-methyladenine (3-MA) were determined by Western blot. RESULTS:Honokiol significantly inhibited the viability of A549 cells in a dose-dependent manner (P<0.05). The number of the intracellular acidic autophageic vacuoles with bright red fluorescence was significantly increased after honokiol treatment. The protein level of LC3-Ⅱ/LC3-I in the A549 cells was significantly enhanced after honokiol (40 μmol/L) treatment, and the ratio of LC3-Ⅱ/LC3-I was significantly decreased by treatment with 3-MA (P<0.05). Furthermore, treatment with honokiol (40 μmol/L) in the A549 cells for 48 h also resulted in significant down-regulation of phosphorylated form of mTOR (P<0.01), while the total protein level was not changed. CONCLUSION:Honokiol significantly inhibits the growth of lung cancer A549 cells and induces the autophagy, which may be correlated with inhibition of mTOR signaling pathway.     

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.