Effects of AZD8055 on autophagy and apoptosis in cholangiocarcinoma cells

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

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.     

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.     

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.     

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.     

Regulation of retinoid X receptor-mediated-autophagy pathway in rat pulmonary ischemia/reperfusion injury

AIM: To investigate the regulation of retinoid X receptor (RXR)-mediated autophagy pathway in rat pulmonary ischemia/reperfusion (I/R) injury. METHODS: The male SPF-grade SD rats were randomly divided into 7 groups (n=10):normal control (C) group, sham (S) group, sham plus 9-cis-retinoic acid (SRA) group, sham plus HX531 (SH) group, I/R group, I/R plus 9-cis-retinoic acid (RA) group and I/R plus HX531 (HX531) group. The model of pulmonary I/R injury was established by clamping the left hilum of lung for 30 min followed by 180 min of reperfusion. The animals in C group didn't receive any treatment. Only sternotomy was performed for the rats in S group, the hilum of lung was not clamped, and the rats were mechanically ventilated for 210 min. The clamping of the left hilum of lung for 30 min followed by 180 min of reperfusion was performed in I/R group. In SRA, SH, RA and HX531 groups, 9-cis-re-tinoic acid (5 mg/kg) was intraperitoneally injected at 90 min before establishment of the model. In SH group and HX531 group, HX531 at 5 mg/kg was intraperitoneally injected at 30 min before establishment of the model. Left lung tissues were removed after 180 min of reperfusion for determing the index of quantitative assessment (IQA) of alveolar damage. The pathological changes of the lung were observed by HE staining, and immunofluorescence staining was used to observe the changes of RXRα in various lung tissues. The mRNA expression of autophagy-associated molecules LC3, beclin 1 and mTOR was detected by RT-PCR. The protein levels LC3-Ⅱ, beclin 1 and p-mTOR in each group were determined by Western blot. RESULTS: Compared with C group, the lung IQA, the mRNA expression of LC3 and beclin 1, and the protein levels of LC3 -Ⅱ and beclin 1 in I/R, RA and HX531 groups were increased significantly, the mTOR mRNA and p-mTOR protein levels were decreased (P<0.05), and the morphological structure of the lung was also impaired. Compared with I/R group, the lung IQA and the expression of LC3 and beclin 1 at mRNA and protein levels were significantly decreased, the mRNA expression of RXRα and mTOR, and the protein level of p-mTOR were increased in RA group (P<0.05), and the structural damage of the lung tissue was also significantly reduced. No statistically significant difference was observed between I/R group and HX531 group. Compared with RA group, the lung IQA and the expression of LC3 and beclin 1 at mRNA and protein levels was increased significantly, the mRNA expression of RXRα and mTOR, and the protein level of p-mTOR were decreased in HX531 group (P<0.05), and the morphological damage of the lung tissue was increased. CONCLUSION: The activation of RXR effectively alleviates the pulmonary I/R injury in rats. The protective role of RXR in lung tissue may be related to the inhibition of autophagy pathway.     

Effects of astragaloside IV combined with ginsenoside Rg1 on autophagy and PI3K signaling pathways of PC12 cells induced by oxygen glucose deprivation/reoxygenation

ATM: To probe the effect and the mechanism of astragaloside IV and ginsenoside Rg1 on autophagy of PC12 cells induced by oxygen glucose deprivation/reoxygenation (OGD/R). METHODS: The autophagy injury model of PC12 cells induced by OGD/R was established(PC12 cells were exposed to 2 h of OGD followed by 24 h of reoxygenation). The effects of astragaloside IV combined with ginsenoside Rg1 on autophagy of PC12 cells were observed, and the mechanism was studied through PI3K Ⅰ/Akt/mTOR and PI3K Ⅲ/becline-1/Bcl-2 signaling pathways. RESULTS: After OGD/R, LC3-Ⅱ/LC3-Ⅰin PC12 cells was increased. Astragaloside IV, ginsenoside Rg1 and astragaloside IV combined with ginsenoside Rg1 restrained the increase in LC3-Ⅱ/LC3-Ⅰ, the effect of the combination was greater than using the drug alone. Ginsenoside Rg1, astragaloside IV combined with ginsenoside Rg1 up-regulated the phosphorylation level of PI3K Ⅰ, Akt and mTOR. The effects of the combination were stronger than those of using the drug alone. Astragaloside IV, astragaloside IV combined with ginsenoside Rg1 inhibited the protein expression of PI3K Ⅲ and becline-1, the effects of the combination were better than those of single astragaloside IV and single ginsenoside Rg1. Meanwhile, the combination treatment increased Bcl-2 protein expression. CONCLUSION: The autophagy of PC12 cells induced by OGD/R is inhibited by astragaloside IV and ginsenoside Rg1. Furthermore, astragaloside IV combined with ginsenoside Rg1 plays synergitic inhibition on autophagy, the mechanism may be related to PI3K Ⅰ/Akt/mTOR and PI3K Ⅲ/becline-1/Bcl-2 signaling pathways.     

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.     

Angiotensin II type 1 receptor autoantibodies induces INS-1 islet β-cell apoptosis by upregulation of autophagy

AIM: To explore whether the angiotensin Ⅱ type 1 receptor autoantibodies (AT1-AA) induces islet β-cell apoptosis and whether autophagy is involved in the process. METHODS: The INS-1 cells treated with AT1-AA at 10^-6 mol/L for 24 h, and then the apoptosis was analyzed by flow cytometry, Western blot and Hoechst 33258 staining. In addition, the expression of autophagy-related proteins such as LC3 and beclin 1 were determined by Western blot. The effects of AT1-AA on the apoptosis, autophagy and viability of INS-1 cells with or without 3-methyladenine (3-MA; a common autophagy inhibitor) or telmisartan (an angiotensin Ⅱ type 1 receptor blocker) pretreatment, were detected by flow cytometry, Western blot and CCK-8 assay. RESULTS: Treatment with AT1-AA at 10^-6 mol/L for 24 h significantly reduced the cell viability (P<0.05). Compared with the negative IgG control group, the apoptotic cells increased after incubation with AT1-AA for 12 h, 24 h and 36 h, respectively (P<0.05). Moreover, the protein levels of LC3 and beclin 1 also increased gradually with the prolongation of treatment time, and the elevation of apoptosis and autophagy were blocked by telmisartan. After pretreatment with 3-MA, the apoptotic rate of the cells was obviously decreased compared with the cells treated with AT1-AA alone. CONCLUSION: AT1-AA induces the apoptosis of INS-1 islet β cells by upregulating autophagy via the angiotensin Ⅱ type 1 receptor pathway.     

Effects of PTEN/AKT/mTOR signalling pathway on regulation of autophagy in renal tissues of diabetic rats

ATM:To observe the expression change of PTEN and autophagy in the renal tissues of diabetic rats, and to explore the regulatory mechanism of PTEN/AKT/mTOR pathway to autophagy in diabetic nephropathy. METHODS: The Sprague-Dawley rats were randomly divided into normal control group and diabetic group (8 in each group). The diabetic rat model was established by injection of streptozotocin. The biochemical and kidney indexes were measured after the model of diabetic nephropathy was successfully established. The expression location of PTEN in the renal tubular epithelial cells was observed by the method of immunohistochemistry. The protein levels of autophagy-related protein LC3, PTEN and PTEN/AKT/mTOR signalling molecules were determined by Western blotting. The mRNA expression of PTEN was detected by real-time PCR. RESULTS: The blood glucose, 24 h urine protein and kidney index in diabetic group were all obviously higher than those in control group. Compared with control group, the protein levels of LC3I and LC3II in diabetic group were obviously decreased. PTEN was mainly located in the renal tubular epithelial cells. Compared with control group, the protein expression of PTEN was significantly down-regulated in diabetic group. Furthermore, the activity of PTEN/AKT/mTOR pathway increased in diabetic nephropathy rats. CONCLUSION: The level of autophagy in renal tissues of diabetic rats is decreased, whereas the activity of PTEN/AKT/mTOR pathway is increased. The level of autophagy may be mediated by PTEN/AKT/mTOR pathway.     

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.     

Role of retinoic acid X receptor in regulation of autophagy in rat alveolar type Ⅱ epithelial cells induced by hypoxia/reoxygenation

AIM To investigate the regulatory effect of retinoic acid X receptor （RXR） on autophagy induced by hypoxia/reoxygenation （H/R） in rat alveolar type Ⅱ epithelial cells （AEC Ⅱ） and its molecular mechanism. METHODS AEC Ⅱ were cultured in normoxia. The cells growing to logarithmic growth phase were randomly divided into 5 groups： （1） control （Con） group： cells were cultured for 30 h under normal operation； （2） H/R group： cells were cultured in hypoxia condition for 6 h and then in reoxygenation condition for 24 h； （3） DMSO group： cells were pretreated 1.5 h with medium containing less than 0.1% DMSO before modeling， and the rest were treated the same as the H/R group； （4） 9-cis-retinoic acid （9-RA） group： cells were pretreated for 1 h with 9-RA （100 nmol/L） before hypoxia； （5） HX531 group： cells were treated with 9-RA （100 nmol/L） for 0.5 h， then treatment with HX531 （2.5 μmol/L） for 1 h. CCK-8 assay was used to detect the cell viability. Immunofluorescence staining was used to observe the expression of RXRα. Transmission electron microscope was used to observe the changes of intracellular ultrastructure， and the mRNA expression of adenosine AMP-activated protein kinase （AMPK）， beclin 1， LC3， mammalian target of rapamycin （mTOR） and P62 was detected by RT-PCR. Western blot was used to detected the protein levels of p-AMPK， beclin 1， LC3-Ⅱ， p-mTOR and P62. RESULTS Compared with Con group， the cell viability in H/R， DMSO， 9-RA and HX531 groups were significantly decreased. The mRNA expression of AMPK， beclin 1 and LC3 was significantly increased， and the protein levels of p-AMPK， beclin 1 and LC3-Ⅱ were also increased. The mRNA expression of mTOR and P62 was decreased， and the protein levels of p-mTOR and P62 were also decreased （P<0.05）. The cell injury in 9-RA group was alleviated and autophagy level was significantly lower than that in H/R， DMSO and HX531 groups （P<0.05）， and no significant difference among H/R， DMSO and HX531 groups was observed （P>0.05）. CONCLUSION H/R induces autophagy of AEC Ⅱ. Activating RXR reduce the damage of AEC Ⅱ cells induced by H/R， and its mechanism may be related to the inhibition of autophagy.     

Antidepressant-like effect of N-palmitoylethanolamide by prefrontal cortex PPARα pathway in a rat model

AIM: To investigate the role of cortical peroxisome proliferator-activated receptor α (PPARα) in the regulation of depression-like behavior in the rats by N-palmitoylethanolamide (PEA). METHODS: A rat model of chronic unpredictable mild stress (CUMS) was established. The rats (n=70) were randomly divided into normal control group, CUMS model group, CUMS+ fluoxetine (10 mg/kg) group, CUMS+ PEA (2.5, 5 and 10 mg/kg) groups and CUMS+ PEA (10 mg/kg)+ MK886 (3 mg/kg) group. On the 8th day during CUMS, the drugs were continuously admi-nistered for 28 d. The body weight and the related behavioral changes in the open-field test and sucrose consumption test were monitored every week. On the 36th day, some of the brain tissues from the rats were fixed in 4% formalin solution for histomorphological and immunohistochemical observations to determine the number and morphological changes of prefrontal cortex (PFC) neurons and the protein expression of synaptophysin (SYP). Other brain tissues were quickly removed, PFC was separated and weighed, and Western blot and RT-PCR were used to detect the expression of PPARα at protein and mRNA levels in the PFC of rats. RESULTS: Compared with CUMS model group, PEA increased the body weight gain, the sucrose preference rate, and the locomotion time and distance in the open-field test, and shortened the immobility time in the open-field test. PEA increased the weight of PFC, the percentage of PFC/brain weight and the number of neurons in PFC, and improved the morphological changs of the neurons. PEA also up-regulated the protein expression of SYP in PFC, and down-regulated the expression of PPARα at mRNA and protein levels in the PFC of CUMS model rats (P<0.05). In addition, compared with PEA (10 mg/kg) group, MK886 significantly reduced the body weight gain of the rats, the percentage of sucrose preference and the locomotion distance in the open-field test, and increased the immobility time in the open-field test on the 35th day during CUMS. The number of neurons SYP expression in PFC tissues were decreased, and the expression of PPARα at protein and mRNA levels was increased in MK886 group. CONCLUSION: PEA may antagonize the depression-like behavior of rats by regulating the PPARα pathway in PFC, improving synaptic plasticity of PFC and protecting the neurons.     

Inhibition of autophgay enhances resveratrol-induced apoptosis of human chondrosarcoma cells

AIM: To investigate whether autophagy is up-regulated when resveratrol (Res) induces apoptosis in chondrosarcoma, and to study the effects of autophagy inhibitor combined with Res on chondrosarcoma. METHODS: SW1353 cells were divided into 4 groups: control group, Res group, 3-methyladenine (3MA) group, and Res+3MA group. Electron microscopy was used to observe the autophagyosomes in control group and Res group. At the same time, the viability of the cells in the 4 groups was detected by CCK-8 assay. TUNEL staining and Western blotting (for determining the levels of cleaved caspase-3, Bax and Bcl-2) were used to reflect levels of apoptosis in all groups. The expression of autophagy-related proteins Beclin 1, LC3-Ⅱ and p62 was detected by Western blotting. RESULTS: Exposure of the cells to Res resulted in a decrease in cell viability and an increase in the level of apoptosis (P<0.05). Compared with control group, the level of apoptosis was increased but the autophagy was decreased (P<0.05). Compared with Res group, the cell viability and the level of autophagy were decreased and the level of apoptosis was increased (P<0.05). CONCLUSION: Resveratrol induces apoptosis and autophagy, and inhibition of autophgay enhances resveratrol-induced apoptosis in chondrosarcoma.     

Inhibitory effect of butylphthalide on apoptosis of hippocampal neurons in Alzheimer disease rats via SIRT1/NF-κB signaling pathway

AIM: To investigate the effect of butylphthalide on apoptosis of hippocampal neurons in Alzheimer disease (AD) rats via SIRT1/NF-κB signaling pathway and its mechanism. METHODS: AD rat model was established by intragastric administration of AlCl₃ and intraperitoneal injection of D-galactose. After treated with butylphthalide at 25 mg/kg (low dose), 50 mg/kg (medium dose) and 100 mg/kg (high dose), the effects of butylphthalide on the morphology of hippocampal neurons, apoptosis rate, and the protein levels of Bcl-2, Bax, cleaved caspase-3 and the SIRT1/NF-κB signaling pathway associated proteins were determined by HE staining, flow cytometry and Western blot, respectively. After treated with SIRT1 agonist SRT1720 and inhibitor sirtinol, the role of SIRT1/NF-κB signaling pathway in hippocampal neuronal apoptosis was observed. On the basis of giving 50 mg/kg butylphthalide, sirtinol was administered, and the effect of butylphthalide on neuronal apoptosis regulated by SIRT1/NF-κB signaling pathway was evaluated. RESULTS: The morphology of hippocampal neurons in the AD rats were improved, the apoptosis rate of hippocampal neurons and the protein levels of Bax and cleaved caspase-3 were inhibited, and the protein levels of Bcl-2 and the activation of SIRT1/NF-κB signaling pathway were promoted by butylphthalide significantly (P<0.05). The protein expression of Bcl-2 and the activation of SIRT1/NF-κB signaling pathway were promoted, and the apoptosis of hippocampal neurons and the protein levels of Bax and cleaved caspase-3 were inhibited by SRT1720 remarkably (P<0.05), whereas the effect of sirtinol was contrary to that of SRT1720. After sirtinol treatment, the inhibitory effect of butylphthalide on apoptosis of hippocampal neurons, the protein levels of Bax and cleaved caspase-3, and the promotion of Bcl-2 protein expression in hippocampal neurons were markedly weakened (P<0.05). CONCLUSION: Butylphthalide inhibits the apoptosis of hippocampal neurons in the AD rats by down-regulating the protein expression of Bax and cleaved caspase-3, and up-regulating the protein expression of Bcl-2 through activating SIRT1/NF-κB signaling pathway.     

Abietic acid alleviates AGEs-induced apoptosis and endoplasmic reticulum stress in cardiomyocytes via inducing autophagy

AIM: This study aims to explore the effect of abietic acid (AA) on advanced glycosylation end products (AGEs)-induced apoptosis and endoplasmic reticulum stress in H9c2 cardiomyocytes. METHODS: H9c2 cells were divided into 5 groups. The cells in control group were treated with saline for 24 h. The cells in AGEs treatment group were treated with AGEs (100 mg/L) for 24 h. The cells in AGEs+AA (10, 25 and 50 μmol/L) groups were simulta-neously treated with AGEs (100 mg/L) and AA (10, 25 and 50 μmol/L) for 24 h. The cell viability was measured by MTT assay. The protein levels of myoglobin (Mb), creatine kinase MB isoenzyme (CK-MB), cardiac troponin I (cTnI), C/EBP homologous protein (CHOP), cleaved caspase-12, GADD34, BiP, LC3, P62 and beclin 1 were determined by Western blot. The levels of lactate dehydrogenase (LDH) were measured by ELASA. The apoptosis was analyzed by flow cytometry. RESULTS: The low concentration (<50 μmol/L) of abietic acid had no obvious effect on the viability of H9c2 cells. The high concentration (>50 μmol/L) of abietic acid decreased the viability of H9c2 cells. The levels of Mb, CK-MB, cTnI and LDH in AGEs group were higher than those in control group (P<0.05). Compared with AGEs group, the levels of Mb, CK-MB, cTnI and LDH in AGEs+AA (10, 25 and 50 μmol/L) groups were obviously reduced (P<0.05). Abietic acid at concentrations of 10, 25 and 50 μmol/L inhibited AGEs-induced apoptosis, elevated the protein levels of CHOP and cleaved caspase-12, and attenuated expression of GADD34 and BiP (P<0.05). Moreover, abietic acid at concentrations of 10, 25 and 50 μmol/L suppressed AGEs-induced decreased ratio of LC3-Ⅱ/LC3-Ⅰ and expression of beclin 1, and enhanced the expression of P62 (P<0.05). 3-Methyladenine, an inhibitor of autophagy, reversed the effect of abietic acid on the protein levels of LC3, Mb, cleaved caspase-12 and BiP (P<0.05). CONCLUSION: Abietic acid alleviates AGEs-induced apoptosis and endoplasmic reticulum stress in H9c2 cardiomyocytes via inducing autophagy.     

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.     

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.     

Cordycepin inhibits proliferation and migration of gallbladder cancer cell SNU-308 by ERK1/2, Ezrin and Akt signaling pathways

AIM: To investigate the effects of cordycepin on the proliferation and migration abilities of gallbladder cancer cell line SNU-308 and its molecular mechanism. METHODS: The viability of SNU-308 cells treated with cordycepin at different concentrations was measured by MTT assay and the colony formation ability was also detected. The effect of cordycepin on apoptosis was analyzed by flow cytometry with Annexin V/PI double staining. The protein levels of apoptosis and autophagy markers, and the phosphorylation level of Akt, ERK1/2 and Ezrin were evaluated by Western blot. Immunofluorescence staining was also used to analyze the expression level of LC3 after cordycepin treatment. Wound healing assay and Transwell assay were performed to evaluate the migration ability of the SNU-308 cells after cordycepin treatment. Wound healing assay was also used to evaluate the effects of Akt inhibitor, ERK1/2 inhibitor and Ezrin knockdown on the changes of migration ability. RESULTS: Cordycepin significantly inhibited the viability and the ability of colony formation of gallbladder cancer cells (P<0.05). Induction of apoptosis by cordycepin were revealed by flow cytometry (P<0.05). The protein expression of Bcl-2 was down-regulated, while the protein levels of Bax, cytochrome C (Cyto C), Fas, FasL and cleaved caspase-3 were increased and the autophagy marker beclin 1 and the ratio of LC3-Ⅱ/I were upregulated by Western blot analysis (P<0.05). LC3 accumulation in the cytoplasm after cordycepin treatment was demonstrated by immunofluorescence staining. Cordycepin treatment resulted in the inhibition of cell migration were detected by Transwell assay and wound healing assay (P<0.05). The protein levels of p-Akt, p-ERK1/2 and p-Ezrin were down-regulated after cordycepin treatment (P<0.05). Besides, Ezrin knockdown, Akti-1/2 and GDC-0994 all resulted in the inhibition of migration ability (P<0.05). CONCLUSION: Cordycepin induces apoptosis and autophagy to inhibit gallbladder can-cer cell proliferation and migration by regulating ERK1/2, Ezrin and Akt signaling pathways.     

Effect of acteoside on behavioral changes and endoplasmic reticulum stress in prefrontal cortex of depressive rats

AIM: To explore the effect of acteoside on behavioral changes and endoplasmic reticulum stress (ERS) in prefrontal cortex of depressive rats. METHODS: Sprague-Dawley (SD) rats (n=108) were randomly divided into 6 groups:control group, model group, fluoxetine (20 mg/kg) group, low-dose (30 mg/kg) acteoside group, medium-dose (60 mg/kg) acteoside group and high-dose (120 mg/kg) acteoside group, with 18 rats in each group. The depressive-like rat model was established by chronic unpredictable mild stress (CUMS) combined with solitary way for 28 d. The rats in fluoxetine group and acteoside groups were treated with fluoxetine (20 mg/kg) or acteoside (30 mg/kg, 60 mg/kg and 120 mg/kg) once daily by intragastric administration for 3 weeks. The rats in control group and model group were both given equal volume of saline by intragastric administration for 3 weeks. The behavioral changes were detected by the open-field test and sugar preference experiment. The protein expression of glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) was assessed by immunofluorescence and Western blot. The caspase-3 activity was measured by spectrophotometer. RESULTS: Compared with control group, the total distance, time spent in the center and sugar intake were all decreased, the expression of GRP78 and CHOP was increased, and the activity of caspase-3 was increased in model group, fluoxetine group and acteoside groups (P<0.05). Compared with model group, the total distance, time spent in the center and sugar intake were increased, the expression of GRP78 and CHOP was reduced, and the activity of caspase-3 was decreased (P<0.05) in fluoxetine group and acteoside groups. CONCLUSION: Acteoside improves depressive-like behaviors in depressive rats, which may be related to the inhibition of ERS and neuronal apoptosis in prefrontal cortex.