Rip2 induces autophagy and its mechanism in human pancreatic cancer cells

AIM:To explore whether receptor-interacting protein 2 (Rip2) induces autophagy and its under-lying mechanisms in human pancreatic cancer cell line Panc-1. METHODS:The empty plasmid pEGFP-C2 or recombinant plasmid pEGFP-Rip2 was transfected into the Panc-1 cells by jetPRIME reagent. The untreated cells served as control group. The protein levels of Rip2, autophagy-related molecules (beclin-1 and LC3-Ⅱ), and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway-related proteins were determined by Western blot 48 h after transfection. The morphological changes of the autophagosomes were observed by transmission electron microscopy. RESULTS:The protein level of Rip2 was significantly increased in the Panc-1 cells transfected with pEGFP-Rip2 plasmid. The protein expression of beclin-1 and LC3-Ⅱ in pEGFP-Rip2 group was higher than that in control group and pEGFP-C2 group (all P<0.01). An increased number of autophagosomes was observed under transmission electron microscope in pEGFP-Rip2 group as compared with control group and pEGFP-C2 group. Furthermore, the protein levels of p-mTOR and p-AKT in pEGFP-Rip2 group were lower than those in control group and pEGFP-C2 group (all P<0.01), while no significant difference of the total mTOR and AKT protein levels was found among the 3 groups. CONCLUSION:Rip2 induces autophagy in the Panc-1 cells and its mechanism may be related to inhibiting the activation of PI3K/Akt/mTOR pathway.     

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 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.     

Roles of PI3K/Akt/mTOR signaling pathway in macrophage autophagy and atherosclerotic plaque instability

AIM：To investigate whether selective inhibition of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway stabilizes the vulnerable atherosclerotic plaques by promoting macrophage autophagy.METHODS：In in vitro study, casodex (20 μmol/L), rapamycin (10 nmol/L) or mTOR-siRNA (30 nmol/L) was used to treat mouse macrophage cell line RAW 264.7. Inflammation-related cytokines secreted by macrophages were measured by means of ELISA. Ultrastructural changes of the macrophages were examined by transmission electron microscopy. The mRNA and protein expression levels of Akt, mTOR and autophage-related protein Beclin 1 were assayed by real-time fluorescence quantitative RT-PCR and Western blotting. The expression of autophagy-related indicator LC3-II was detected by immunofluorescence and Western blotting. In in vivo study, 24 New Zealand white rabbits underwent balloon-induced abdominal aortic wall injury and were fed with a diet of 1% cholesterol for 8 weeks. The rabbits were randomly divided into control group (n=8), casodex group (1.0 mg·kg-1·d-1, n=8) and rapamycin group (0.5 mg·kg-1·d-1, n=8). Four weeks after drug administration, intravascular ultrasound (IVUS) was carried out to observe the plaque imaging. Ultrastructural changes of the macrophages and the protein expression of Akt, mTOR and LC3-II in the macrophages were also measured.RESULTS：In in vitro study, more typical autophagosomes were detected in casodex-, rapamycin- or mTOR-siRNA-treated cells. The expression level of LC3-II increased, but Beclin 1,p-Akt and p-mTOR significantly decreased in the 3 treatment groups. The concentration of IL-10 decreased while IFN-γ significantly increased in the treatment groups. In in vivo study, IVUS found that external elastic membrane area (EEMA),plaque area(PA) and plaque burden (PB) significantly decreased in casodex and rapamycin treatment groups. Expression of LC3-II increased significantly in the 2 treatment groups. The staining of RAM-11 and p-mTOR in the macrophages was significantly reduced as compared with control group.CONCLUSION：Selective inhibition of PI3K/Akt/mTOR signaling pathway reduces the infiltration of macrophages and stabilizes the vulnerable atherosclerotic plaques by promoting macrophage autophagy.     

Effects of NOD8 on H2O2-induced apoptosis in human hepatocytes

AIM: To observe the effects of NOD8 on H2O2-induced apoptosis in human L02 hepatocytes. METHODS: pEGFP-C2 and pEGFP-NOD8 plasmids were transfected into L02 cells by JetPRIME, respectively. The apoptosis of these transfected cells was induced by H2O2. The cells were divided into pEGFP-C2 group, pEGFP-C2+H2O2 group and pEGFP-NOD8+H2O2 group. MTT assay was used to detect the cell viability. NOD8 protein expression was determined by Western blotting. The cell apoptosis was observed by Hoechst 33342 staining and apoptotic rate was evaluated by flow cytometry. The caspase-3 activity was analyzed by a colorimetric method. RESULTS: L02 cells were stimulated by H2O2 at concentrations of 0.2～2 mmol/L for 6 h, and H2O2 at concentration of 1 mmol/L was chosen to induce apoptosis determined by MTT assay. The protein expression of NOD8 significantly increased in the cells transfected with pEGFP-NOD8 plasmid. More cellular nucleus with strong blue fluorescence by Hoechst 33342 staining in pEGFP-C2+ H2O2 group were observed, indicating that apoptosis was increased, while the apoptosis in pEGFP-NOD8+H2O2 group significantly reduced. The apoptotic rate in pEGFP-C2+H2O2 group was obviously increased, whereas that in pEGFP-NOD8+H2O2 group was significantly decreased. The caspase-3 activity in pEGFP-C2+H2O2 group was remarkably increased. By contrast, the activity of caspase-3 was significantly reduced in pEGFP-NOD8+H2O2 group.CONCLUSION: NOD8 inhibits H2O2-induced apoptosis in L02 cells and the mechanism may be related to inhibition of caspase-3 activity.     

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

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

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.     

NVP-BEZ235 induces autophagy of polycystic kidney rat cholangiocytes

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

LPS regulates macrophage autophagy through PI3K/Akt/mTOR pathway

AIM: To detect the activation of macrophage autophagy caused by lipopolysaccharide (LPS) and the possible related signaling pathways. METHODS: The macrophage cell line RAW264.7 cultured in vitro was divided into 5 groups according to the culture environment, including normal culture group, starvation-activated sautophagy group, LPS group, LPS+PI3K inhibitor (hVps34) group and LPS+mTOR inhibitor (rapamycin) group. Fluorescent expression vector pcDNA3.1-GFP-LC3 constructed in previous work was transfected into the macrophages.The fluorescence microscopy was used to detect the formation of autophagosome. The mRNA expression of autophagy-associated genes Atg5, Atg7, LC3-II and Bnip3 in the macrophages was detected by qRT-PCR. The protein levels of LC3-II, p-Akt and p-mTOR were determined by Western blotting, so as to evaluate the molecular pathways of autophagy in LPS-activated macrophages. RESULTS: The macrophages stably expressing GFP-LC3 were successfully established, which were used to observe the autophagy under fluorescence microscope.Compared with normal culture group, the autophagy in starvation group, LPS+hVps34 group and LPS+rapamycin group was significantly increased. The mRNA expression levels of Atg5, LC3-II and Bnip3 were significantly increased in starvation group, LPS+hVps34 group and LPS+rapamycin group, while in LPS group, those decreased slightly. The protein level of p-Akt in starvation group, LPS group and LPS+rapamycin group was significantly increased, while p-mTOR in starvation group, LPS+hVps34 group and LPS+rapamycin group significantly declined. LC3-II expression level in starvation group, LPS+hVps34 group and LPS+rapamycin group was higher than that in control group and LPS group. CONCLUSION: LPS regulates macrophage autophagy, and its possible pathway is the PI3K/Akt/mTOR pathway, but there are some other effective regulatory pathways.     

Effect of NOD8 on production of nitric oxide,IL-1β and TNF-α in LPS-treated macrophages

AIM: To investigate the effect of NOD8 on lipopolysaccharide (LPS)-induced releases of nitric oxide (NO), tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β) in RAW264.7 cells. METHODS: The plasmids of pEGFP-C2 and pEGFP-NOD8 were transfected into RAW264.7 cells respectively. The transfected and non-transfected cells were stimulated by LPS for 0, 6, 12 and 24 h. NO production was evaluated by Griess reagent assay, and the levels of IL-1β and TNF-α were measured by ELISA. The protein expression of NOD8 and the nuclear translocation of nuclear factor κB (NF-κB) p65 subunit were detected by Western blotting. The level of activated caspase-1 was determined by fluorimetric method. RESULTS: Compared with pEGFP-C2 group, the protein expression of NOD8 was significantly elevated in pEGFP-NOD8+LPS group. The releases of NO, IL-1β and TNF-α were obviously increased after RAW264.7 cells were treated with LPS for 6 h, 12 h and 24 h, and while the secretion of NO was significantly reduced in the cells transfected with pEGFP-NOD8 and induced by LPS for 12 h and 24 h, and the release of IL-1β was also significantly reduced at 6 h, 12 h and 24 h. However, no significant difference of TNF-α release was observed between pEGFP-C2+LPS group and pEGFP-NOD8+LPS group. The activation of caspase-1 in RAW264.7 cells stimulated with LPS for 6 h, 12 h and 24 h was markedly increased, and the expression of NF-κB p65 subunit in the cytoplasm was significantly decreased, indicating that p65 nuclear translocation was increased. In addition, the activation of caspase-1 and the nuclear translocation of p65 were significantly inhibited in pEGFP-NOD8+LPS group. CONCLUSION: NOD8 suppresses the releases of LPS-induced NO and IL-1β in RAW264.7 cells by inhibiting the activation of caspase-1 and NF-κB.     

Irbesartan alleviates hepatic steatosis in db/db mice by inducing auto-phagy

AIM: To investigate the effect of irbesartan on the fatty liver of db/db mice and whether autophagy is involved in the process. METHODS: Male db/db mice (n=24) were randomly divided into model group and irbesartan group, and 12 db/m mice with similar age and weight were selected as normal control group. After 16 weeks of intervention respectively, the fatty liver-related parameters including body weight, liver index, blood lipid, liver function and pathological changes in the liver were observed. The protein levels of p-PI3K, p-Akt, and p-mTOR, as well as Atg-7, beclin-1 and LC3B in the liver tissues were detected by Western blot, and the autophagosomes in the liver were observed under electron microscope. RESULTS: Compared with the model group, the body weight, liver index, blood lipids, alanine and aspartate aminotransferase were decreased in irbesartan group (P<0.05). Moreover, the pathological changes in the liver were significantly ameliorated in irbesartan group than that of model group. Importantly, the protein levels of p-PI3K, p-Akt and p-mTOR were decreased with irbesartan administration, while the expression of Atg-7, beclin-1 and LC3B-Ⅱ was increased(P<0.05), which resulted in a distinct increase in autophagosomes. CONCLUSION: Irbesartan alleviates hepatic steatosis in db/db mice by inhibiting the PI3K/Akt/mTOR signaling pathway and upregulating the protein expression of Atg-7, beclin-1 and LC3B-Ⅱ, thereby inducing autophagy in hepatocytes.     

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.     

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.     

Notch1 promotes renal tubulointerstitial fibrosis in renal tissues of diabetic mice by inhibiting autophagy through Akt/mTOR signaling pathway

AIM: To observe the changes of Notch1 expression and autophagy in the renal tissues of diabetic mice, and to explore the regulatory effect of Notch1 on tubulointerstitial fibrosis by inhibiting autophagy in diabetic nephro-pathy. METHODS: The mice were randomly divided into normal control group (db/m mice) and diabetes group (db/db mice), with 8 rats in each group. After 12 weeks of feeding, the mice were sacrificed and the corresponding biochemical indexes were measured. The protein expression of Notch1 in the renal tubular epithelial cells was observed by immunohistochemical staining. The protein levels of Notch1, PTEN, p-Akt (Thr308), Akt, p-mTOR (Ser2448), mTOR, LC3, P62, collagen type Ⅰ (Col-Ⅰ) and collagen type Ⅲ (Col-Ⅲ) were determined by Western blot. RESULTS: Compared with the db/m mice, the blood glucose, glycosylated hemoglobin, serum creatinine, triglyceride and total cholesterol were increased in the db/db mice (P<0.01). Renal tubular epithelial cell vacuolar degeneration, renal tubular expansion and interstitial inflammatory cell infiltration in db/db mouse renal tissues with HE staining were observed. The images of Masson staining showed collagenous fiber-like substance deposition in the glomerular capillaries and renal interstitium, and disarrangement of tubular structure in the renal tissues of db/db mice. The protein expression levels of PTEN and LC3-Ⅱ were decreased (P<0.01 or P<0.05), while the protein levels of Notch1, P62, p-mTOR (Ser2448), p-Akt (Thr308), Col-I and Col-III were increased in the db/db mice as compared with the db/m mice (P<0.01). However, no significant change of total mTOR and Akt proteins between the 2 groups was found. CONCLUSION: Notch1 protein expression was increased, PTEN expression was significantly reduced, Akt/mTOR pathway was activated, autophagy was inhibited, and fibrosis was aggravated in the renal tissues of the diabetic mice.     

PI3K/Akt signaling pathway regulates autophagy induced by acute kidney injury in septic rats

AIM：To investigate the autophagy induced by sepsis and acute kidney injury, and the regulation of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway in this process. METHODS：The rats were subjected to cecal ligation and puncture (CLP) or sham operation. Histopathologic changes of the renal tissues were examined by HE staining. Blood urea nitrogen (BUN) and serum creatinine (SCr) were measured by chemical colorimetry. The protein expression of microtubule-associated protein light chain 3 I/II (LC3 I/II), beclin-1 and p-Akt at different time points after CLP was detected by Western blotting. In vitro, human proximal tubular epithelial cell line HK-2 were treated with LPS to induce autophagy. The protein expression of LC3 I/II and p-Akt in the HK-2 cells after LPS treatment at different time points and different concentrations was detected by Western blotting. These molecules in HK-2 cells and apoptosis of HK-2 cells treated with LPS plus PI3K inhibitor or Akt inhibitor were also detected. RESULTS：Compared with sham group, the severe changes of renal histopathological injuries in CLP groups were observed, the levels of BUN and SCr in CLP groups were significantly increased. LC3 I/II, beclin-1 and phosphorylation of Akt gradually increased after CLP. After treatment with LPS, the expression of p-Akt (308) in the HK-2 cells gradually increased in a dose- and time-dependent fashion. The expression of beclin-1 and p-Akt (472) reached a peak at 8 h or 10 mg/L LPS treatment. Treatment with PI3K or Akt inhibitor down-regulated the expression of LC3 and promoted the apoptosis of HK-2 cells. CONCLUSION：Autophagy in the kidney is induced by sepsis and acute kidney injury. PI3/Akt signaling pathway may be involved in this process.     

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.     

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.     

Adiponectin inhibits decrease in autophagy of rat H9c2 cardiomyocytes induced by β1-adrenergic receptor autoantibodies

AIMTo investigate whether adiponectin inhibits the decrease in autophagy of rat H9c2 cardiomy?ocytes induced by β₁-adrenergic receptor (β₁-AR) autoantibodies (β₁-AA), and to explore its mechanism. METH?ODS: SD rats were actively immunized with β₁-AR extracellular second loop (β₁-AR-ECII) antigen peptide. Affinity chromatography was used to purify β₁-AA in serum of the SD rats. The viability of H9c2 cells was measured by CCK-8 assay. The mRNA levels of LC3B and beclin-1 in the H9c2 cells were detected by real-time PCR. The protein levels of LC3-II, P62, AMP-activated protein kinase (AMPK) and phosphorylated AMPK (p-AMPK) were determined by Western blot. RESULTSPretreatment with adiponectin at 10 μg/L for 1 h reversed the decreased viability of H9c2 cells induced by β₁-AA. Compared with control group, β₁-AA decreased the mRNA expression of LC3B and beclin-1, decreased the protein level of LC3-II, and increased the expression of P62 protein in the H9c2 cells, suggesting that β₁-AA decreased the autophagic flux in cardiomyocytes. Adiponectin obviously reversed β₁-AA-induced decline in autophagic flux, and up-regulated the phosphorylation level of AMPK decreased by β₁-AA. Treatment with AMPK inhibitor Compound C for 30 min, we observed that the mRNA expression of LC3B and beclin-1 and the protein level of LC3-II in the H9c2 cells decreased by β₁-AA were not effectively reversed by adiponectin, but the increase in P62 protein expression was still effectively reversed, indicating that adiponectin increased autophagosome production dependent on the AMPK pathway, but increased autophagosome clearance independent on the AMPK pathway. CONCLUSION Adiponectin inhibits the decreased autophagy of H9c2 cardiomyocytes induced by β₁-AA.     

Inhibitory effect of ischemic postconditioning on autophagy induced by focal cerebral ischemia reperfusion in rats

AIM: To investigate the effect of ischemic postconditioning (IPC) on autophagy induced by focal cerebral ischemia reperfusion (I/R) in rats. METHODS: Healthy male SD rats were assigned randomly into sham-operation (sham) group, I/R group and IPC group with 10 rats in each group. The rats in sham group were only exposed the right common, internal and external carotid artery surgically. The rats in I/R group were subjected to right middle cerebral artery occlusion (MCAO) by the modified Longa suture method for 2 h followed by 24 h of reperfusion. The rats in IPC group were subjected to MCAO for 2 h followed by reperfusion of the ipsilateral common carotid artery occlusion for 10 s for 5 episodes, and then reperfusion for 24 h. Autophagy was obeserved by transmission electron microscopy (TEM). The protein levels of mammalian target of rapamycin (mTOR), p-mTOR and microtubule associated protein light chain 3 (LC3)-II in brain tissue of the rats were determined by Western blot. Pathological changes of brain tissue were observed by HE staining. RESULTS: The protein levels of mTOR and p-mTOR in IPC group were significantly higher than those in I/R group (P<0.05). The expression of LC3-II in IPC group was significantly lower than that in I/R group (P<0.01). The cerebral infarction area and brain water content in IPC group were significantly lower than those in I/R group (P<0.01). HE staining showed that neurons degeneration and necrosis in IPC group were significantly alleviated compared with I/R group. TEM observation showed that IPC revealed fewer autophagosomes, with much less severe cell damage than that in I/R group. CONCLUSION: IPC reduces brain ischemia reperfusion damage by decreasing autophagy of brain cells, which might be related to the activation of mTOR.     

Baicalein induces autophagy in breast cancer cells

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