Up-regulation of miR-181a attenuates releases of CSE-induced pro-inflammatory factors and expression of collagen IV,fibronectin and α-SMA in human bronchial epithelial cells

AIM: To investigate the effect and potential mechanism of microRNA-181a (miR-181a) on cigarette smoke extract (CSE)-induced the productions of pro-inflammatory factors and the expression of collagen IV, fibronectin and α-smooth muscle actin (α-SMA) in human bronchial epithelial cells (HBECs). METHODS: CSE-induced miR-181a expression was detected by RT-qPCR in the HBECs. After tansfected with miR-181a mimic, the releases of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6 and transforming growth factor-β1 (TGF-β1) were measured by ELISA, the protein expression of collagen IV, fibronectin and α-SMA was determined by Western blot. The activation of NF-κB/TGF-β1/Smad3 pathway was also evaluated by Western blot. RESULTS: CSE increased the levels of TNF-α, IL-1β, IL-6 and TGF-β1 and the expression of collagen IV, fibronectin and α-SMA, and decreased the expression of miR-181a in the HBECs (P<0.05). However, transfected with miR-181a mimic partially prevented the releases of TNF-α, IL-1β, IL-6 and TGF-β1, and inhibited the expression of collagen IV, fibronectin and α-SMA (P<0.05). Additionally, the activation of NF-κB/TGF-β1/Smad3 evoked by CSE was attenuated after transfected with miR-181a mimic. CONCLUSION: Up-regulation of miR-181a prevents the releases of CSE-induced pro-inflammatory factors and expression of collagen IV, fibronectin and α-SMA in the HBECs, and its mechanism may be related to the inhibition of NF-κB/TGF-β1/Smad3 pathway.     

Effect of microRNA-132 transfection on lipopolysaccharide-induced inflammation in rat alveolar macrophages

AIM: To investigate the effect of microRNA-132 (miR-132) transfection on the lipopolysaccharide (LPS)-induced inflammation in rat alveolar macrophages. METHODS: The rat alveolar macrophage NR8383 cultured without pyrogen in vitrowere divided into blank control group, negative control group and transfected group. The cells in the 3 groups were transfected with phosphate buffer solution (PBS), Lipofectamine 2000 and synthesized miR-132 mimic respectively. The cell proliferation was detected by Cell Counting Kit-8 (CCK-8) assay. Real-time PCR was used to detect the expression of miR-132 in the cells. After NR8383 cells were stimulated with LPS for 6 h, the NF-κB DNA-binding activity was measured by electrophoretic mobility shift assay (EMSA). The expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in NR8383 cells was assayed by Western blotting.RESULTS: After transfection, the expression of miR-132 was significantly higher than that in blank control group and negative control group. The growth of NR8383 cells in transfected group was significantly inhibited compared with blank control group and negative control group (P<0.05). After the cells were stimulated with LPS, the productions of NF-κB, TNF-α and IL-6 in transfected NR8383 cells were decreased compared with blank control group and negative control group (P<0.05).CONCLUSION: Transfection of alveolar macrophages with miR-132 significantly suppresses the cell growth, and inhibits inflammatory responses induced by LPS.     

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.     

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.     

Relationship between rat hepatic fibrosis and regulation of hepatic stellate cell autophagy by microRNA-181a

AIM To observe the changes of liver structure, the levels of transforming growth factor-β1 （TGF-β1）, microRNA-181a, LC3-II/-I, beclin-1 and collagen deposition in hepatic fibrosis （HF） rats induced by carbon tetrachloride （CCl₄）, and the effect of microRNA-181a on autophagy of rat hepatic stellate cells （HSCs） induced by TGF-β1, and to explore the possible mechanism of microRNA-181a in regulating HSC activation and HF. METHODS Wistar rats （n=40） were randomly divided into 5 groups （with 8 in each）： control group （subcutaneous injection of olive oil, 3 mL/kg, twice a week）, and CCl₄-induced HF groups of 2, 4, 6 and 8 weeks （subcutaneous injection of 40% CCl₄, 3 mL/kg, twice a week for 2, 4, 6 and 8 weeks, respectively）. Masson staining was used to evaluate the changes of HF in rats. The levels of TGF-β1 in serum and liver tissue of the rats were measured by ELISA. The level of microRNA-181a in rat liver tissues was detected by RT-qPCR. The protein levels of LC3-II/-I, beclin-1, α-smooth muscle actin （α-SMA）, collagen type I （Col I） and collagen type Ⅲ （Col Ⅲ） in rat liver tissues were measured by Western blot. HSC-T6 cells were transfected with microRNA-181a inhibitor, or pretreated with the autophagy inhibitor 3-methyladenine （3-MA）, before treatment with TGF-β1 to stimulate autophagy. The expression of microRNA-181a, LC3-II/-I, beclin-1, α-SMA, Col I and Col Ⅲ in HSC-T6 cells were determined by RT-qPCR and Western blot. RESULTS The levels of TGF-β1, microRNA-181a, LC3-II/-I ratio and beclin-1 in liver tissues showed an overall trend of increasing with the progression of HF, and microRNA-181a expression showed a positive correlation with autophagy-associated proteins （P<0.01）. MicroRNA-181a level was significantly increased, which was associated with TGF-β1-induced autophagy and activation of HSC-T6 cells.MicroRNA-181a expression was significantly down-regulated in the HSC-T6 cells transfected with microRNA-181a inhibitor, along with suppression of autophagy and cell activation （P<0.01）, which were similar to the effects of 3-MA treatment. CONCLUSION CCl₄ promotes rat HF, the microRNA-181a expression of liver tissue, and autophagy in a time-dependent manner. Reducing the expression of microRNA-181a in HSC-T6 cells inhibits the autophagy of HSCs-T6 cells induced by TGF-β1. The regulation of HSC autophagy by microRNA-181a may be involved in rat HF.     

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 cigarette smoking condensate on HDAC2 and inflammatory mediators in mouse myoblast C2C12 cells

AIM: To investigate the effect of cigarette smoking condensate on histone deacetylase 2 (HDAC2) and inflammatory mediators in mouse myoblast C2C12 cells. METHODS: C2C12 cells were treated with cigarette smoke extract (CSE). HDAC2 siRNA was transfected into the cells using Lipofectamine^TM 2000. The levels of interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) in the culture supernatants were measured by ELISA, and the expression of HDAC2 at mRNA and protein levels was determined by real-time PCR and Western blotting. RESULTS: The expression of HDAC2 at mRNA and protein levels in CSE group was lower than that in control group (P<0.05). The supernatant levels of IL-8 and TNF-α in CSE group were significantly higher than those in control group (P<0.05). When the cells were transfected with HDAC2 siRNA followed by CSE stimulation, the expression of HDAC2 at mRNA and protein levels was decreased, and the supernatant levels of IL-8 and TNF-α were significantly increased as compared with CSE group and control group (P<0.05).CONCLUSION: Under the oxidative stress condition, C2C12 cells generate high levels of IL-8 and TNF-α by down-regulating the expression of HDAC2.     

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.     

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.     

Effects of miR-155 over-expression on expression of inflammatory factors and indolamine 2, 3-dioxygenase in microglial BV-2 cells

AIM:To explore the effect of microRNA-155(miR-155)over-expression on the expression of inflammatory factors and indolamine 2, 3- dioxygenase (IDO) in the microglial BV-2 cells. METHODS:For over-expression of miR-155, the BV-2 cells were transfected with lentiviral vector carrying mmu-miR-155. The expression of inflammatory factors was detected by cytometric bead array system (CBA). The mRNA expression of inflammatory factors and IDO was analyzed by real-time PCR. The protein levels of suppressor of cytokine signalling 1 (SOCS1), p-p38 MAPK and IDO were determined by Western blot. RESULTS:The expression of miR-155 was up-regulated in the BV-2 cells transfected with lentiviral vector carrying mmu-miR-155 compared with LPS treatment group (P<0.01). The miR-155 over-expression promoted the secretion of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), monocyte chemotactic protein-1 (MCP-1) and IL-10, and inhibited the secretion of IL-12. The miR-155 over-expression increased the mRNA expression of IL-6, TNF-α, IL-10 and IDO, also increased the protein levels of IDO and p-p38 MAPK, but decreased the protein expression of SOCS1 (P<0.01). LPS promoted the secretion of IL-6, TNF-α, MCP-1 and IL-12, also increased the mRNA expression of IL-6, TNF-α and IDO, meanwhile, increased the protein levels of IDO, p-p38 MAPK and SOCS1 (P<0.01). CONCLUSION:Over-expression of miR-155 promotes the secretion of related imflammatory factors and protein expression of IDO in microglial BV-2 cells mediated with SOCS1 and p38 MAPK signaling pathway.     

miR-451 inhibits inflammatory responses in glomerular mesangial cells by targeting Psmb8 in diabetic nephropathy mice

AIM: To investigate the effect of microRNA (miR)-451 by targeting proteasome subunit β type 8 (Psmb8) on the inflammatory responses in mouse glomerular mesangial cells (MCs) under high-and low-glucose conditions. METHODS: The expression levels of miR-451, IL-18 mRNA and TNF-α mRNA were detected by qPCR. The protein expression levels of IL-18, TNF-α and Psmb8 were determined by Western blot when miR-451 was over-expressed and down-expressed in the MCs. Moreover, the expression of IL-18 and TNF-α was detected when Psmb8 was silenced by si-Psmb8 in MCs. RESULTS: The expression of miR-451 was significantly decreased in the MCs treated with high glucose compared with low glucose group (P<0.01). However, the expression of Psmb8 was increased in high glucose group as compared with low glucose group (P<0.01). Moreover, the expression levels of Psmb8, IL-18 and TNF-α were significantly decreased when miR-451 was over-expressed in high glucose group (P<0.01). Additionally, the expression levels of IL-18 and TNF-α were significantly reduced when Psmb8 was silenced in the MCs under high glucose condition. CONCLUSION: miR-451 reduces the expression of inflammatory factors via targeting Psmb8 in the MCs under high glucose condition. Therefore, miR-451 may play a role in inflammation of diabetic nephropathy.     

Role of NOX1 in TNF-α-induced oxidative damage and inflammation in alveolar epithelial cells

AIM: To explore the role of NADPH oxidase 1 (NOX1) in tumor necrosis factor-α (TNF-α)-induced oxidative damage and inflammation in alveolar epithelial cells.METHODS: The mRNA and protein expression levels of NOX1 in alveolar epithelial cells after TNF-α treatment were determined by real-time PCR and Western blot. NOX1 siRNA and its negative control were transfected into the alveolar epithelial cells. After the induction of TNF-α, NOX1 levels in the cells were measured by real-time PCR and Western blot, and the content of malondialdehyde (MDA) in the cells was detected by thiobarbituric acid method. Xanthine oxidation assay was used to detect the activity of superoxide dismutase (SOD) in the cells. The contents of interleukin-4 (IL-4), IL-6 and IL-1β in cell culture medium were examined by ELISA. The rate of apoptosis was analyzed by flow cytometry. Western blot was used to detect the level of apoptotic protein cleaved caspase-3.RESULTS: The expression of NOX1 at mRNA and protein levels in TNF-α-induced cells was increased after induction (P<0.05). After transfection of NOX1 siRNA, the expression of NOX1 at mRNA and protein levels in the cell was downregulated (P<0.05). Transfection of siRNA negative control had no effect on the expression level of NOX1 in the cells. The content of MDA in the cells after TNF-α treatment was increased, the activity of SOD was reduced, the releases of IL-4, IL-6 and IL-1β by the cells were increased, and the apoptotic rate and the level of apoptotic protein cleaved caspase-3 were increased as compared with the cells that were not treated with TNF-α (P<0.05). The content of MDA in the cells with NOX1 knockdown induced by TNF-α was reduced, the activity of SOD elevated, and the releases IL-4, IL-6 and IL-1β, the apoptotic rate and the level of apoptotic protein cleaved caspase-3 decreased, as compared with the cells only treated with TNF-α induction (P<0.05).CONCLUSION: TNF-α induces the expression of NOX1 in the alveolar epithelial cells. Knockdown of NOX1 expression reduces cellular oxidative damage, releases of inflammatory factors, and cell apoptosis.     

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.     

Effects of rosiglitazone on expression of SOCS1/SOCS3 and inflammatory reaction in RAW 264.7 cell-derived foam cells

AIM: To investigate whether perioxisome proliferator-activated receptor γ (PPARγ) ligand rosiglitazone regulates suppressor of cytokine signaling 1 (SOCS1) and SOCS3 expression as well as pro-inflammatory/anti-inflammatory responses in RAW 264.7 cell-derived foam cells. METHODS: The concentrations of TNF-α, IL-6 and IL-10 in the cultured supernatant of RAW 264.7 cell-derived foam cells were detected by ELISA, and the ratios of TNF-α/IL-10 and IL-6/IL-10 were calculated. RT-PCR and Western blotting were used to analyze the effects of rosiglitazone on the expression of SOCS1 and SOCS3 at mRNA and protein levels. RESULTS: The concentrations of TNF-α, IL-6 and IL-10, and ratios of TNF-α/IL-10 and IL-6/IL-10 in foam cell group were obviously higher than those in control group, but the concentrations of the above factors in oxidized low-density lipoprotein (ox-LDL) +rosiglitazone group were apparently lower than those in foam cell group. The expression of SOCS1 and SOCS3 at mRNA and protein levels in oxLDL+rosiglitazone group was apparently higher than that in control and foam cell group. CONCLUSION: PPARγ ligand rosiglitazone up-regulates the expression of SOCS1 and SOCS3 at mRNA and protein levels and regulates the balance of pro-inflammatory/anti-inflammatory responses in RAW 264.7 cell-derived foam cells.     

Effect of autophagic inhibitor 3-methyladenine on apoptosis of hippo-campal neurons induced by Aβ(25-35)

AIM: To investigate the influence of autophagy on the apoptosis of hippocampal neurons in the rat model of Alzheimer disease.METHODS: Sprague-Dawley rats were divided into model group, autophagic inhibitior 3-methyladenine (3-MA) pretreatment group and control group.In model group, the rats were anesthetized and placed in a stereotaxic apparatus.Hippocampus CA1 area microinjection was performed and Aβ(25-35) was applied to establish the model of AD.3-MA in 0.9% saline was administered by the same way prior to Aβ(25-35) infusion.The learning and memory ability of the rats was observed by Morris water maze.The ultrastructure of the hippocampal neurons, the formation of autophagic vesicles, beclin-1 expression and cell apoptosis were detected after behavioral experiment.RESULTS: Compared with model group, the learning and memory ability of the rats in 3-MA group significantly impaired (P<0.05) and the apoptotic rate of the hippocampal neurons significantly increased (P<0.05).Moreover, the expression of beclin-1 was declined.In model group, hippocampal neurons showed double membrane wrapped in the autophagic vacuoles, and the neuronal damages were significantly milder than that in 3-MA group.CONCLUSION: Decrease in the levels of neuronal autophagy increases the neuronal apoptosis, indicating that increasing neuronal autophagy may have therapeutic potential for AD.     

Effects of bFGF on brain edema,nerve function injury and autophagy related protein in rats with traumatic brain injury

AIM:To study the effects of basic fibroblast growth factor (bFGF) on brain edema, nerve function damage and autophagy related proteins in rats with head injury. METHODS:The rat model of craniocerebral injury (CI) was constructed. The rats were divided into control group, CI group, and low-, middle-and high-dose bFGF groups (n=10). The CI model was established in CI group, while the rats in control group were not given epidural impact. The rats in low-dose, middle-dose and high-dose bFGF groups were given bFGF at 2, 4 and 6 μg, respectively, by intraperitoneal injection after 30 min. The neurological function in the rats was evaluated by improved neurological function scoring. The rat brain tissues were taken, and the water content was detected. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1β in the brain tissue were measured by ELISA. The malondialdehyde (MDA) content was analyzed by thiobarbituric acid method. The activity of superoxide dismutase (SOD) was examined by WST-8 assay. The glutathine peroxidase (GSH-Px) activity was detected by colorimetric method. The protein levels of autophagy related proteins LC3-Ⅱ and beclin-1 in the brain tissues were determined by Western blot. RESULTS:The neurological function score was increased significantly of the rats in CI group. The rat model of craniocerebral injury was successfully constructed. Neurological function scores in the rats in low-dose, middle-dose and high-dose bFGF groups were reduced, the water content of the brain tissue was also reduced (P<0.05). The levels of TNF-α, IL-6 and IL-1 β were decreased in the brain tissues (P<0.05), the content of MDA was declined (P<0.05), the activities of SOD and GSH-Px were increased (P<0.05), the protein levels of LC3-Ⅱ and beclin-1 were decreased, compared with the untreated rats in CI group (P<0.05). CONCLUSION:bFGF improves the nerve function of the rats with craniocerebral injury, reduces the water content of the brain tissue, reduces the expression of autophagic protein LC3-Ⅱ and beclin-1.The mechanism is related to the inhibition of inflammatory reaction and oxidative damage.     

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.