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.     

NOD8 inhibits autophagy in human pancreatic cancer cells and effect of apoptosis on autophagy regulatd by NOD8

AIM: To explore whether NOD8 inhibits autophagy in human pancreatic cancer cells and its underlying mechanisms, and to investigate the effect of apoptosis on the autophagy regulated by NOD8. METHODS: The empty plasmid pEGFP-C2 and recombinant plasmid pEGFP-NOD8 were transfected into the Panc-1 cells using JetPRIME reagent.The untransfected cells served as control group. The protein levels of NOD8, autophagy-related proteins beclin-1 and LC3-II, and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway-related proteins Akt, p-Akt, mTOR and p-mTOR were determined by Western blot 48 h after transfection. Meanwhile, the number of LC3 spots was quantified by immunofluorescence staining. Furthermore, after a broad caspase inhibitor Z-VAD-FMK was applied to NOD8-over-expressing cells, the protein expression levels of beclin-1 and LC3-II were detected by Western blot and the number of LC3 spots was observed by immunofluorescence staining. RESULTS: The protein level of NOD8 in pEGFP-NOD8 group was significantly higher than that in control group and pEGFP-C2 group (P<0.01). The protein expression of beclin-1 and LC3-II, and the number of LC3 spots in pEGFP-NOD8 group were significantly decreased as compared with control group and pEGFP-C2 group. Moreover, the protein levels of p-AKT and p-mTOR in pEGFP-NOD8 group were higher than those in control group and pEGFP-C2 group, while no significant difference of mTOR and AKT protein expression was found among these 3 groups. Furthermore, the protein levels of beclin-1 and LC3-II, and the number of LC3 spots in pEGFP-NOD8+Z-VAD-FMK group were significantly increased compared with pEGFP-NOD8 group. CONCLUSION: NOD8 inhibits autophagy in the Panc-1 cells and its mechanism may be related to the activation of PI3K/Akt/mTOR pathways. Apoptosis enhances the inhibitory effect of NOD8 on autophagy.     

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.     

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.     

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.     

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

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

Effect of miR-22 secreted by macrophage exosomes on cardiomyocyte autophagy under uremic toxin stimulation

AIM To investigate the effect of microRNA-22 (miR-22) secreted by macrophage exosomes on the autophagy of H9c2 cardiomyocytes under uremic toxin stimulation. METHODS The macrophage-derived exosomes stimulated by indoxyl sulfate (IS) were collected and co-cultured with H9c2 cells. The levels of miR-22 in the macrophages, macrophage-derived exosomes and H9c2 cells were detected by RT-qPCR. The viability of H9c2 cells was measured by CCK-8 assay. The expression of exosome surface marker protein CD63 and autophagy-related proteins LC3 and P62 was determined by Western blot. RESULTS Under IS stimulation, the expression of exosome surface marker protein CD63 in the macrophages was significantly higher than that in control group (P<0.05), and the levels of miR-22 in the macrophages and macrophage-derived exosomes were significantly increased (P<0.01). With the increase in macrophage exosome concentration, the viability of H9c2 cells was decreased gradually (P<0.05), and the stimulation of macrophage exosomes reduced P62 expression and promoted the conversion of LC3-I to LC3-II in a dose-dependent manner (P<0.05). Macrophage-derived exosomes increased the ratio of LC3-II to LC3-I but decreased P62 protein expression in the H9c2 cells transfected with miR-22 mimic compared with the cells transfected with corresponding negative control miRNAs (P<0.05). However, miR-22 inhibitor yielded contrasting results. CONCLUSION IS-stimulated macrophages increase expression of miR-22 in cardiomyocytes through exosomes, and promote autophagy of the cardiomyocytes.     

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.     

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.     

Lipophagy inhibits foam cell formation by reducing cholesterol content

AIM To observe the changes of lipophagy during foam cells formation, and to determine the effect of lipophagy on the lipid content and cholesterol outflow of foam cells. METHODS Human THP-1 monocytes were induced by phorbol-12-myristate-13-acetate for 48 h to differentiate into macrophages, and then were incubated with 50 mg/L oxidized low-density lipoprotein （oxLDL） to form foam cells. Lipids in foam cell were stained by oil red O, and the lipid content was determined. The total cholesterol （TC） and free cholesterol （FC） levels in foam cells were measured by cholesterol testing kit. Cholesteryl ester （CE） and CE/TC ratio were calculated. The cholesterol efflux rate was detected by cholesterol efflux assay kit. The expression of autophagy-related proteins, including autophagy-related protein 5 （Atg5）, microtubule-associated protein 1 light chain 3 （LC3） and P62, were detected by Western blot. The colocalization of lipid droplets （LD） and LC3 was detected by immunofluorescence staining. The autophagy inducer rapamycin （Rap） or blocker 3-methyladenine （3MA） was used to intervene foam cells, and the expression of Atg5, LC3 and P62, the co-expression of LD and LC3, the cholesterol content and the cholesterol efflux rate were determined. RESULTS Formation of foam cells was observed at 24 h after stimulation with oxLDL at 50 mg/L, as indicated by intracellular CE/TC ratio exceeding 50%.Cholesterol efflux assay revealed that the cholesterol efflux rate increased within 24 h during foam cell formation but decreased after 48 h （P<0.05）. Western blot results displayed that the expression of Atg5 and LC3-II/LC3-I ratio were increased within 24 h of foam cell formation, but was deceased after 48 h （P<0.05）. The expression of P62 was decreased within 24 h but was increased at 48 h （P<0.05）. The colocalization of LD and LC3 was increased at 24 h but was decreased at 48 h after oxLDL stimulation. Treatment with Rap up-regulated the expression of Atg5 and LC3-II/LC3-I ratio, reduced the level of P62, increased the colocalization of LD and LC3, promoted the cholesterol efflux, anf reduced cholesterol content in foam cells （P<0.05）. On the contrary, 3MA inhibited the expression of Atg5, reduced LC3-II/LC3-I ratio, elevated the level of P62, decreased the colocalization of LD and LC3, reduced the outflow of cholesterol, increased the content of TC and CE, and elevated CE/TC ratio in foam cells （P<0.05）. CONCLUSION Lipophagy is enhanced at 24 h but decreased at 48 h during foam cell formation. Lipophagy inhibited foam cell formation by reducing cholesterol content and increasing cholesterol efflux.     

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 continuous renal replacement therapy on mRNA expression of autophagy-related molecules and prognosis in patients with acute kidney injury

AIM To explore the effect of continuous renal replacement therapy （CRRT） on mRNA expression of autophagy-related molecules and the prognosis in the patients with acute kidney injury （AKI）. METHODS A total of 174 patients from our hospital who were diagnosed to have AKI and underwent CRRT between February 2015 and March 2018 were involved in this study. The plasma levels of interleukin-1β （IL-1β） and interleukin-6 （IL-6）, the serum creatinine （SCr） level, and the mRNA expression levels of autophagy-related molecules, including microtubule-associated protein 1 light chain 3-II （LC3-II）, autophagy-related protein 5 （Atg5） and beclin-1, in the monocytes from peripheral blood were compared before and after CRRT. According to the survival of AKI patients after 4 weeks of CRRT, the enrolled patients were divided into death group （n=43） and survival group （n=131）, and the mRNA expression levels of the above molecules were compared between the 2 groups. RESULTS After CRRT treatment, the plasma levels of IL-1β and IL-6, the level of SCr, and the mRNA expression levels of LC3-II, Atg5 and beclin-1 in the monocytes were significantly lower than those before CRRT treatment （P<0.05）. The mRNA expression levels of LC3-II, Atg5 and beclin-1 in death group were significantly higher than those in survival group （P<0.05）. The positive correlation between SCr and IL-1β, IL-6, LC3-II or beclin-1 was observed （P<0.05）, and no correlation between SCr and Atg5 was found （P>0.05）. CONCLUSION CRRT decreases the mRNA expression levels of autophagy-related molecules in the patients with AKI and reduces the autophagy activity, which is protective for the patients. These autophagy-related molecules may be applied as a potential markers to predict the prognosis of CRRT.     

Astragalosides inhibit autophagy and apoptosis of rat cardiomyocytes induced by H2O2 through mTOR signaling pathway

AIM: To investigate the effects of astragalosides on autophagy and apoptosis of rat cardiomyocytes induced by hydrogenperoxide (H₂O₂).METHODS: The injury model of H9c2 cells induced by H₂O₂ was established, and the cells in astragalosides group and rapamycin group were treated with 20 mg/L astragalosides and 0.1 mg/L rapamycin, respectively. The apoptotic rate was detected by flow cytometry. The autophagy was observed by acridine orange staining. Western blot was used to detect the protein levels of p-mTOR, P70S6K, LC3 and caspase-3. RESULTS: Compared with H₂O₂ group and rapamycin group, the viability of H9c2 cells in astragalosides group was significantly increased (P<0.05). The shape of the H9c2 cells in astragalosides group was complete, the nuclei were stained with yellow-green fluorescence, and the chromatin was distributed evenly. The protein levels of p-mTOR and P70S6K in the H9c2 cells of astragalosides group were significantly increased (P<0.05), whereas the protein levels of LC3, cleaved caspase-3 and caspase-3 in the H9c2 cells of astragalosides group were decreased significantly (P<0.05). CONCLUSION: Astragalosides enhance the viability, inhibit the apoptosis, increase the protein levels of p-mTOR and P70S6K, and decrease the protein levels of LC3, cleaved caspase-3 and caspase-3 in the H₂O₂-induced rat myocardial H9c2 cells. The mechanism is related to the mTOR signaling pathway.     

CD147 affects autophagy of prostate cancer PC-3 cells

AIM：To study the autophagy of prostate cancer PC-3 cells induced by CD147 in vitro. ME-THODS：The method of amino acid starvation to induce autophagy was used. The expression of CD147was detected by Western blotting. To study the functional effects of CD147 on autophagy in prostate cancer PC-3 cells, the down-regulation of CD147expression was induced by the technique of RNAi. The conversion of autophagic marker protein LC3-I to LC3-II was determined by Western blotting. The cell death after starvation-induced autophagy was analyzed by trypan blue exclusion assay. RESULTS：The CD147 expression gradually increased in starvation-induced autophagy. The down-regulation of CD147 significantly increased the expression of autophagy-related protein LC3-II compared with control group. Meanwhile, the cell death rates increased from (19.3±3.1)% and (22.3±3.5)% in control groups to (38.4±3.1)% in silencing the expression of CD147in the PC-3 cells (P<0.05). CONCLUSION：CD147 inhibits starvation-induced autophgy and autophagy death in the prostate cancer PC-3 cells.     

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．     

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.     

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.     

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.     

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.