Autophagy-dependent and -independent involvement of AMP-activated protein kinase in 6-hydroxydopamine toxicity to SH-SY5Y neuroblastoma cells

The role of the main intracellular energy sensor adenosine monophosphate (AMP)-activated protein kinase (AMPK) in the induction of autophagic response and cell death was investigated in SH-SY5Y human neuroblastoma cells exposed to the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA). The induction of autophagy in SH-SY5Y cells was demonstrated by acridine orange staining of intracellular acidic vesicles, the presence of autophagosome- and autophagolysosome-like vesicles confirmed by transmission electron microscopy, as well as by microtubule-associated protein 1 light-chain 3 (LC3) conversion and p62 degradation detected by immunoblotting. 6-OHDA induced phosphorylation of AMPK and its target Raptor, followed by the dephosphorylation of the major autophagy inhibitor mammalian target of rapamycin (mTOR) and its substrate p70S6 kinase (S6K). 6-OHDA treatment failed to suppress mTOR/S6K phosphorylation and to increase LC3 conversion, p62 degradation and cytoplasmatic acidification in neuroblastoma cells in which AMPK expression was downregulated by RNA interference. Transfection of SH-SY5Y cells with AMPK or LC3β shRNA, as well as treatment with pharmacological autophagy inhibitors suppressed, while mTOR inhibitor rapamycin potentiated 6-OHDA-induced oxidative stress and apoptotic cell death. 6-OHDA induced phosphorylation of p38 mitogen-activated protein (MAP) kinase in an AMPK-dependent manner, and pharmacological inhibition of p38 MAP kinase reduced neurotoxicity, but not AMPK activation and autophagy triggered by 6-OHDA. Finally, the antioxidant N-acetyl cysteine antagonized 6-OHDA-induced activation of AMPK, p38 and autophagy. These data suggest that oxidative stress-mediated AMPK/mTOR-dependent autophagy and AMPK/p38-dependent apoptosis could be valid therapeutic targets for neuroprotection.     

Autophagy induction by xanthoangelol exhibits anti‐metastatic activities in hepatocellular carcinoma

Xanthoangelol (XAG), a prenylated chalcone isolated from the Japanese herb Angelica keiskei Koidzumi, has been reported to exhibit antineoplastic properties. However, the specific anti‐tumor activity of XAG in human hepatocellular carcinoma (HCC), and the relevant mechanisms are not known. Herein, we evaluated the effect of XAG against HCC in vitro and in vivo. Although XAG treatment did not significantly reduce the viability of the Hep3B and Huh7 cell lines, it suppressed cell migration, invasion, and EMT. This anti‐metastatic effect of XAG was due to induction of autophagy, because treatment with the autophagy inhibitor 3‐methyadenine (3‐MA) or knockdown of the pro‐autophagy Beclin‐1 effectively abrogated the XAG‐induced suppression of metastasis. Mechanistically, XAG induced autophagy via activation of the AMPK/mTOR signaling pathway, and XAG treatment dramatically increased the expression of p‐AMPK while decreasing p‐mTOR expression. In addition, blocking AMPK/mTOR axis with compound C abrogated the autophagy‐mediated inhibition of metastasis. The murine model of HCC metastasis also showed that XAG effectively reduced the number of metastatic pulmonary nodules. Taken together, our results revealed that autophagy via the activation of AMPK/mTOR pathway is essential for the anti‐metastatic effect of XAG against HCC. These findings not only contribute to our understanding of the anti‐tumor activity of XAG but also provide a basis for its clinical application in HCC. Before this study, evidence of XAG on HCC was purely anecdotal; present study provides the first comprehensive assessments of XAG on HCC metastasis and investigates its underlying mechanism. Results suggest that XAG exerts anti‐metastatic properties against HCC through inducing autophagy which is mediated by the activation of AMPK/mTOR signaling pathway. This research extends our knowledge about the antineoplastic properties of XAG and suggests that induction autophagy may represent future treatment strategies for metastatic HCC.     

Protective Role of AMP-Activated Protein Kinase-Evoked Autophagy on an In Vitro Model of Ischemia/Reperfusion-Induced Renal Tubular Cell Injury

Ischemia/reperfusion (I/R) injury is a common cause of injury to target organs such as brain, heart, and kidneys. Renal injury from I/R, which may occur in renal transplantation, surgery, trauma, or sepsis, is known to be an important cause of acute kidney injury. The detailed molecular mechanism of renal I/R injury is still not fully clear. Here, we investigate the role of AMP-activated protein kinase (AMPK)-evoked autophagy in the renal proximal tubular cell death in an in vitro I/R injury model. To mimic in vivo renal I/R injury, LLC-PK1 cells, a renal tubular cell line derived from pig kidney, were treated with antimycin A and 2-deoxyglucose to mimic ischemia injury followed by reperfusion with growth medium. This I/R injury model markedly induced apoptosis and autophagy in LLC-PK1 cells in a time-dependent manner. Autophagy inhibitor 3-methyladenine (3MA) significantly enhanced I/R injury-induced apoptosis. I/R could also up-regulate the phosphorylation of AMPK and down-regulate the phosphorylation of mammalian target of rapamycin (mTOR). Cells transfected with small hairpin RNA (shRNA) for AMPK significantly increased the phosphorylation of mTOR as well as decreased the induction of autophagy followed by enhancing cell apoptosis during I/R. Moreover, the mTOR inhibitor RAD001 significantly enhanced autophagy and attenuated cell apoptosis during I/R. Taken together, these findings suggest that autophagy induction protects renal tubular cell injury via an AMPK-regulated mTOR pathway in an in vitro I/R injury model. AMPK-evoked autophagy may be as a potential target for therapeutic intervention in I/R renal injury.     

Acetylshikonin induces autophagy‐dependent apoptosis through the key LKB1‐AMPK and PI3K/Akt‐regulated mTOR signalling pathways in HL‐60 cells

Acetylshikonin (ASK) is a natural naphthoquinone derivative of traditional Chinese medicine Lithospermum erythrorhyzon. It has been reported that ASK has bactericidal, anti‐inflammatory and antitumour effects. However, whether ASK induces apoptosis and autophagy in acute myeloid leukaemia (AML) cells and the underlying mechanism are still unclear. Here, we explored the roles of apoptosis and autophagy in ASK‐induced cell death and the potential molecular mechanisms in human AML HL‐60 cells. The results demonstrated that ASK remarkably inhibited the cell proliferation, viability and induced apoptosis in HL‐60 cells through the mitochondrial pathway, and ASK promoted cell cycle arrest in the S‐phase. In addition, the increased formation of autophagosomes, the turnover from light chain 3B (LC3B) I to LC3B II and decrease of P62 suggested the induction of autophagy by ASK. Furthermore, ASK significantly decreased PI3K, phospho‐Akt and p‐p70S6K expression, while enhanced phospho‐AMP‐activated protein kinase (AMPK) and phospho‐liver kinase B1(LKB1) expression. The suppression of ASK‐induced the conversion from LC3B I to LC3B II caused by the application of inhibitors of AMPK (compound C) demonstrated that ASK‐induced autophagy depends on the LKB1/AMPK pathway. These data suggested that the autophagy induced by ASK were dependent on the activation of LKB1/AMPK signalling and suppression of PI3K/Akt/mTOR pathways. The cleavage of the apoptosis‐related markers caspase‐3 and caspase‐9 and the activity of caspase‐3 induced by ASK were markedly reduced by inhibitor of AMPK (compound C), an autophagy inhibitor 3‐methyladenine (3‐MA) and another autophagy inhibitor chloroquine (CQ). Taken together, our data reveal that ASK‐induced HL‐60 cell apoptosis is dependent on the activation of autophagy via the LKB1/AMPK and PI3K/Akt‐regulated mTOR signalling pathways.     

Development of a enterovirus diagnostic assay system for diagnosis of viral myocarditis in humans

The coxsackieviruses type B3 (CVB3) are members of the genus Enterovirus of the family Picornaviridae. They are the commonest cause of chronic myocarditis and dilated cardiomyopathy. However, there is still no effective method for diagnosing CVB3 infection in humans. Here, a fast and accurate system that uses a capsid‐protein‐specific peptide sequence to detect CVB3 in the sera of patients with viral myocarditis was established. The peptide sequence was selected from the whole CVB3 capsid protein sequence by computationally predicting fragments with high antigenicity and low hydrophobicity. Two of eight possible peptide sequences were selected and commercially synthesized. The synthesized peptides encoded either the VP2 or VP1 capsid protein and induced immunoglobulin G antibody expression in immunized rabbits. Anti‐VP2 and anti‐VP1 sera detected the viral proteins extracted from CVB3‐infected HeLa cells. The newly synthesized peptides successfully induced antibody production. These peptides, applied in an ELISA system, detected anti‐CVB3 antibodies in virus‐infected mouse serum. Moreover, an ELISA system based on the VP2 peptide detected CVB3 infection in patients with positively identified CVB3‐induced fulminant myocarditis. These results indicate that these new peptides specifically interact with anti‐CVB3 IgG antibodies in mouse and human sera. This ELISA system should be useful for the clinical diagnosis of enterovirus‐induced myocarditis.     

MiR‐19a Affects Hepatocyte Autophagy via Regulating lncRNA NBR2 and AMPK/PPARα in D‐GalN/Lipopolysaccharide‐Stimulated Hepatocytes

This study aims to evaluate the potential involvement and regulatory mechanism of miR‐19a in hepatocytes autophagy of acute liver failure (ALF). The in vitro hepatocytes injury model of primary hepatocyte and hepatocytes line HL‐7702 was established by D‐galactosamine (D‐GalN) and lipopolysaccharide (LPS) co‐treatment. Relative expression level of miR‐19a and NBR2 was determined by qRT‐PCR. Protein expression of AMPK/PPARα and autophagy‐related gene was determined by Western blot. In hepatic tissue of 20 ALF patients and D‐GalN/LPS‐stimulated hepatocytes, miR‐19a was upregulated and NBR2 was downregulated. D‐GalN/LPS stimulation caused the inactivation of AMPK/PPARα signaling and the decrease of autophagy‐related LC3‐II/LC3‐I ratio and beclin‐1 expression in hepatocytes. The expression of both AMPK/PPARα and NBR2 were negatively controlled by miR‐19a overexpression or knockdown. Moreover, both NBR2 and PPARα were targeted regulated by miR‐19a according to luciferase reporter assay. In D‐GalN/LPS‐stimulated hepatocytes, AMPK activation promoted PPARα expression. AMPK inactivation inhibited the pro‐autophagy effect of miR‐19a and caused the decrease of LC3‐II/LC3‐I ratio and beclin‐1 expression. PPARα activation abrogated the anti‐autophagy effect of miR‐19a mimic and caused the increase of LC3‐II/LC3‐I ratio and beclin‐1 expression. NBR2 knockdown reversed the anti‐autophagy impact of miR‐19a inhibitor and caused the decrease of LC3‐II/LC3‐I ratio and beclin‐1 expression. In summary, our data suggested that miR‐19a negatively controlled the autophagy of hepatocytes attenuated in D‐GalN/LPS‐stimulated hepatocytes via regulating NBR2 and AMPK/PPARα signaling. J. Cell. Biochem. 119: 358–365, 2018. © 2017 Wiley Periodicals, Inc.     

Calpain regulates CVB3 induced viral myocarditis by promoting autophagic flux upon infection

Calpains are calcium-activated neutral cysteine proteases. The dysregulation of calpain activity has been found to be related to cardiovascular diseases, for which calpain inhibition is used as a treatment. Viral myocarditis (VMC) is primarily caused by Coxsackievirus group B3 virus infection (CVB3). CVB3 virus infection induces autophagy and hijacks this process to facilitate its replication. In this study, we found that calpain was significantly activated in hearts affected by VMC. However, pharmacologically inhibiting calpain aggravated VMC symptoms in mice due to myocardial inflammation and cardiac dysfunction. The inhibition of calpain activity in vitro led to the accumulation of LC3-II and increased levels of p62/SQSTM1 protein expression, suggesting that autophagic flux was impaired by calpain inhibition. These effects of calpain inhibition were also observed in capn4-specific myocardial knockout mice in vivo. Furthermore, our results provided evidence that calpain inhibition in VMC, unlike other cardiovascular diseases, exacerbated the disease symptom by impairing CVB3-induced autophagic flux, which may subsequently reduce virus autolysosome degradation. Our findings indicated that calpain inhibition may not be a good treatment for VMC disease in a clinical setting.     

Monotropein promotes angiogenesis and inhibits oxidative stress‐induced autophagy in endothelial progenitor cells to accelerate wound healing

Attenuating oxidative stress‐induced damage and promoting endothelial progenitor cell (EPC) differentiation are critical for ischaemic injuries. We suggested monotropein (Mtp), a bioactive constituent used in traditional Chinese medicine, can inhibit oxidative stress‐induced mitochondrial dysfunction and stimulate bone marrow‐derived EPC (BM‐EPC) differentiation. Results showed Mtp significantly elevated migration and tube formation of BM‐EPCs and prevented tert‐butyl hydroperoxide (TBHP)‐induced programmed cell death through apoptosis and autophagy by reducing intracellular reactive oxygen species release and restoring mitochondrial membrane potential, which may be mediated viamTOR/p70S6K/4EBP1 and AMPK phosphorylation. Moreover, Mtp accelerated wound healing in rats, as indicated by reduced healing times, decreased macrophage infiltration and increased blood vessel formation. In summary, Mtp promoted mobilization and differentiation of BM‐EPCs and protected against apoptosis and autophagy by suppressing the AMPK/mTOR pathway, improving wound healing in vivo. This study revealed that Mtp is a potential therapeutic for endothelial injury‐related wounds.     

Mammalian target of rapamycin inhibition attenuates myocardial ischaemia–reperfusion injury in hypertrophic heart

Pathological cardiac hypertrophy aggravated myocardial infarction and is causally related to autophagy dysfunction and increased oxidative stress. Rapamycin is an inhibitor of serine/threonine kinase mammalian target of rapamycin (mTOR) involved in the regulation of autophagy as well as oxidative/nitrative stress. Here, we demonstrated that rapamycin ameliorates myocardial ischaemia reperfusion injury by rescuing the defective cytoprotective mechanisms in hypertrophic heart. Our results showed that chronic rapamycin treatment markedly reduced the phosphorylated mTOR and ribosomal protein S6 expression, but not Akt in both normal and aortic‐banded mice. Moreover, chronic rapamycin treatment significantly mitigated TAC‐induced autophagy dysfunction demonstrated by prompted Beclin‐1 activation, elevated LC3‐II/LC3‐I ratio and increased autophagosome abundance. Most importantly, we found that MI/R‐induced myocardial injury was markedly reduced by rapamycin treatment manifested by the inhibition of myocardial apoptosis, the reduction of myocardial infarct size and the improvement of cardiac function in hypertrophic heart. Mechanically, rapamycin reduced the MI/R‐induced iNOS/gp91^phox protein expression and decreased the generation of NO and superoxide, as well as the cytotoxic peroxynitrite. Moreover, rapamycin significantly mitigated MI/R‐induced endoplasmic reticulum stress and mitochondrial impairment demonstrated by reduced Caspase‐12 activity, inhibited CHOP activation, decreased cytoplasmic Cyto‐C release and preserved intact mitochondria. In addition, inhibition of mTOR also enhanced the phosphorylated ERK and eNOS, and inactivated GSK3β, a pivotal downstream target of Akt and ERK signallings. Taken together, these results suggest that mTOR signalling protects against MI/R injury through autophagy induction and ERK‐mediated antioxidative and anti‐nitrative stress in mice with hypertrophic myocardium.     

Inhibition of mitochondrial autophagy protects donor lungs for lung transplantation against ischaemia‐reperfusion injury in rats via the mTOR pathway

Impaired mitochondrial function is a key factor attributing to lung ischaemia‐reperfusion (IR) injury, which contributes to major post‐transplant complications. Thus, the current study was performed to investigate the role of mitochondrial autophagy in lung I/R injury and the involvement of the mTOR pathway. We established rat models of orthotopic left lung transplantation to investigate the role of mitochondrial autophagy in I/R injury following lung transplantation. Next, we treated the donor lungs with 3‐MA and Rapamycin to evaluate mitochondrial autophagy, lung function and cell apoptosis with different time intervals of cold ischaemia preservation and reperfusion. In addition, mitochondrial autophagy, and cell proliferation and apoptosis of pulmonary microvascular endothelial cells (PMVECs) exposed to hypoxia‐reoxygenation (H/R) were monitored after 3‐MA administration or Rapamycin treatment. The cell apoptosis could be inhibited by mitochondrial autophagy at the beginning of lung ischaemia, but was rendered out of control when mitochondrial autophagy reached normal levels. After I/R of donor lung, the mitochondrial autophagy was increased until 6 hours after reperfusion and then gradually decreased. The elevation of mitochondrial autophagy was accompanied by promoted apoptosis, aggravated lung injury and deteriorated lung function. Moreover, the suppression of mitochondrial autophagy by 3‐MA inhibited cell apoptosis of donor lung to alleviate I/R‐induced lung injury as well as inhibited H/R‐induced PMVEC apoptosis, and enhanced its proliferation. Finally, mTOR pathway participated in I/R‐ and H/R‐mediated mitochondrial autophagy in regulation of cell apoptosis. Inhibition of I/R‐induced mitochondrial autophagy alleviated lung injury via the mTOR pathway, suggesting a potential therapeutic strategy for lung I/R injury.     

Corilagin inhibits breast cancer growth via reactive oxygen species‐dependent apoptosis and autophagy

Corilagin is a component of Phyllanthus urinaria extract and has been found of possessing anti‐inflammatory, anti‐oxidative, and anti‐tumour properties in clinic treatments. However, the underlying mechanisms in anti‐cancer particularly of its induction of cell death in human breast cancer remain undefined. Our research found that corilagin‐induced apoptotic and autophagic cell death depending on reactive oxygen species (ROS) in human breast cancer cell, and it occurred in human breast cancer cell (MCF‐7) only comparing with normal cells. The expression of procaspase‐8, procaspase‐3, PARP, Bcl‐2 and procaspase‐9 was down‐regulated while caspase‐8, cleaved PARP, caspase‐9 and Bax were up‐regulated after corilagin treatment, indicating apoptosis mediated by extrinsic and mitochondrial pathways occurred in MCF‐7 cell. Meanwhile, autophagy mediated by suppressing Akt/mTOR/p70S6K pathway was detected with an increase in autophagic vacuoles and LC3‐II conversion. More significantly, inhibition of autophagy by chloroquine diphosphate salt (CQ) remarkably enhanced apoptosis, while the caspase inhibitor z‐VAD‐fmk failed in affecting autophagy, suggesting that corilagin‐induced autophagy functioned as a survival mechanism in MCF‐7 cells. In addition, corilagin induced intracellular reactive oxygen species (ROS) generation, when reduced by ROS scavenger NAC, apoptosis and autophagy were both down‐regulated. Nevertheless, in SK‐BR3 cell which expressed RIP3, necroptosis inhibitor Nec‐1 could not alleviate cell death induced by corilagin, indicating necroptosis was not triggered. Subcutaneous tumour growth in nude mice was attenuated by corilagin, consisting with the results in vitro. These results imply that corilagin inhibits cancer cell proliferation through inducing apoptosis and autophagy which regulated by ROS release.     

LY294002 and Rapamycin promote coxsackievirus-induced cytopathic effect and apoptosis via inhibition of PI3K/AKT/mTOR signaling pathway

Coxsackievirus B3 (CVB3) is a common human pathogen for acute myocarditis, pancreatitis, non-septic meningitis, and encephalitis; it induces a direct cytopathic effect (CPE) and apoptosis on infected cells. The Phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT/PKB)/mammalian target of Rapamycin (mTOR) signaling pathway regulates several cellular processes and it is one of the most important pathways in human networks. However, the effect and mechanism of PI3K/AKT/mTOR signaling pathway in CVB3 infected cells are poorly understood. In this study, we demonstrate that inhibition of PI3K/AKT/mTOR signaling pathway increased CVB3-induced CPE and apoptosis in HeLa cells. The activity of downstream targets of PI3K and mTOR is attenuated after CVB3 infection and inhibitors of PI3K and mTOR made their activity to decrease more significantly. We further show that LY294002 and Rapamycin, the inhibitor of PI3K and mTOR respectively, promote CVB3-induced CPE and apoptosis. Taken together, these data illustrate a new and imperative role for PI3K/AKT/mTOR signaling in CVB3 infection in HeLa cells and suggest an useful approach for the therapy of CVB3 infection.     

Kaempferol Mediated AMPK/mTOR Signal Pathway Has a Protective Effect on Cerebral Ischemic-Reperfusion Injury in Rats by Inducing Autophagy

Ischemia/reperfusion (I/R) caused by ischemic stroke treatments leads to brain injury and its pathological mechanism is related to autophagy. The underlying mechanism of kaempferol on cerebral I/R injury needs to be explored. To establish I/R injury, we used a middle cerebral artery occlusion-reperfusion (MCAO) model in rats. MCAO rats were treated with the same amount of saline (I/R group); Treatment group rats were treated orally with kaempferol (50, 100, 200 mg/kg) for 7 days before surgery. After reperfusion for 24 h, the scores of neurological deficits and infarct volume in each group were evaluated. LC3, Beclin-1 p62, AMPK and mTOR protein expression levels were examined by TTC staining, immunofluorescence staining, qRT-PCR and western blotting assay. H&E and TTC staining showed that compared with model group, the infarction size of rats in kaempferol group was markedly reduced. Meanwhile, the results showed that kaempferol had a dose-dependent nerve function repairability. Nissl and TUNEL staining showed that kaempferol could reduce neuronal apoptosis and ameliorate neuronal impairment after I/R. Western blotting and qRT-PCR results showed that kaempferol could protect the brain from ischemia reperfusion by activating autophagy. In addition, add AMPK inhibitor, western blotting and immumohistochemical staining showed that kaempferol mediated AMPK/mTOR signal pathway in MCAO rats. Kaempferol could mediate the AMPK signal pathway to regulate autophagy and inhibit apoptosis to protect brain against I/R injury.

     

Restoration of autophagy by puerarin in ethanol-treated hepatocytes via the activation of AMP-activated protein kinase

We investigated the effects of puerarin, the major isoflavone in Kudzu roots, on the regulation of autophagy in ethanol-treated hepatocytes. Incubation in ethanol (100 mM) for 24 h reduced cell viability by 20% and increased the cellular concentrations of cholesterol and triglycerides by 40% and 20%, respectively. Puerarin stimulation significantly recovered cell viability and reduced cellular lipid accumulation to a level comparable to that in untreated control cells. Ethanol incubation reduced autophagy significantly as assessed by microtubule-associated protein1 light chain 3 (LC3) expression using immunohistochemistry and immunoblot analysis. The reduced expression of LC3 was restored by puerarin in a dose-dependent manner in ethanol-treated cells. The effect of puerarin on mammalian targets of rapamycin (mTOR), a key regulator of autophagy, was examined in ethanol-treated hepatocytes. Immunoblotting revealed that puerarin significantly induced the phosphorylation of 5′AMP-activated protein kinase (AMPK), thereby suppressing the mTOR target proteins S6 ribosomal protein and 4E-binding protein 1. These data suggest that puerarin restored the viability of cells and reduced lipid accumulation in ethanol-treated hepatocytes by activating autophagy via AMPK/mTOR-mediated signaling.     

Ginsenoside Rg1 inhibits autophagy in H9c2 cardiomyocytes exposed to hypoxia/reoxygenation

Ginsenoside Rg1 promotes antioxidative protection and intracellular calcium homeostasis in cardiomyocytes hypoxia/reoxygenation (H/R) model. However, the pharmacological effects of G-Rg1 on autophagy in cardiomyocytes have not been reported. In this study, we employed H9c2 cardiomyocytes as a model to investigate the effects of G-Rg1 on autophagy in cardiomyocytes under H/R stress. Our results showed that H/R induced increased level of LC3B-2, an autophagy marker, in a time-dependent manner in association with decreased cell viability and cellular ATP content. H/R-induced autophagy and apoptosis were further confirmed by morphological examination. 100 μmol/l Rg1-inhibited H/R induced autophagy and apoptosis, and this was associated with the increase of cellular ATP content and the relief of oxidative stress in the cells. Mechanistically, we found that Rg1 inhibited the activation of AMPKα, promoted the activation of mTOR, and decreased the levels of LC3B-2 and Beclin-1. In conclusion, our data suggest that H/R induces autophagy in H9c2 cells leading to cell injury. Rg1 inhibits autophagosomal formation and apoptosis in the cells, which may be beneficial to the survival of cardiomyocytes under H/R.     

雌激素通过GPR30-AMPK-mTOR通路诱导Ishikawa细胞自噬增强细胞活力

雌激素是子宫内膜癌发生发展的重要诱导因子,但关于其在子宫内膜癌中的作用机制目前仍不明确。自噬对细胞的存活具有重要的调节作用,研究发现其在子宫内膜癌发生发展的过程中起重要的调节作用。本文通过探讨雌激素对子宫内膜癌细胞自噬的影响,深入地了解雌激素促进子宫内膜发展的机制,并明确GPR30-MPK-mTOR 通路在其中的作用。MTT及透视电镜的结果显示,雌激素可以诱导细胞的自噬及增强细胞的活力,而这种作用具有一定的时间及浓度依赖性。同时,蛋白质印迹及实时定量PCR结果显示雌激素可以促进LC3、p-AMPK的表达,并且抑制P62、p-mTOR的表达,表明雌激素可以激活AMPK/mTOR通路。沉默G蛋白偶联受体30（GPR30）后,结果显示雌激素诱导细胞的自噬及细胞活力的作用被逆转,并且可以抑制AMPK/mTOR通路的激活,而G-1结果与之相反,表明雌激素通过GPR30激活AMPK/mTOR通路,诱导自噬及细胞活力。此外,加入AMPK抑制剂compound C,可以抑制雌激素诱导细胞的自噬及细胞活力的能力,并且促进P62、p-mTOR表达,降低LC3及p-AMPK表达,表明雌激素通过激活AMPK/mTOR激活细胞自噬及增强细胞活力。同时细胞预先加入自噬抑制剂3-MA或转染ATG5siRNA,可以降低雌激素增强细胞的活力,表明雌激素通过诱导自噬增强细胞活力。综合以上结果,雌激素通过GPR30-AMPK-mTOR通路诱导细胞的自噬增强细胞的活力。     

Danshensu alleviates cardiac ischaemia/reperfusion injury by inhibiting autophagy and apoptosis via activation of mTOR signalling

The traditional Chinese medicine Danshensu (DSS) has a protective effect on cardiac ischaemia/reperfusion (I/R) injury. However, the molecular mechanisms underlying the DSS action remain undefined. We investigated the potential role of DSS in autophagy and apoptosis using cardiac I/R injury models of cardiomyocytes and isolated rat hearts. Cultured neonatal rat cardiomyocytes were subjected to 6 hrs of hypoxia followed by 18 hrs of reoxygenation to induce cell damage. The isolated rat hearts were used to perform global ischaemia for 30 min., followed by 60 min. reperfusion. Ischaemia/reperfusion injury decreased the haemodynamic parameters on cardiac function, damaged cardiomyocytes or even caused cell death. Pre‐treatment of DSS significantly improved cell survival and protected against I/R‐induced deterioration of cardiac function. The improved cell survival upon DSS treatment was associated with activation of mammalian target of rapamycin (mTOR) (as manifested by increased phosphorylation of S6K and S6), which was accompanied with attenuated autophagy flux and decreased expression of autophagy‐ and apoptosis‐related proteins (including p62, LC3‐II, Beclin‐1, Bax, and Caspase‐3) at both protein and mRNA levels. These results suggest that alleviation of cardiac I/R injury by pre‐treatment with DSS may be attributable to inhibiting excessive autophagy and apoptosis through mTOR activation.     

TAK1 activates AMPK‐dependent cytoprotective autophagy in TRAIL‐treated epithelial cells

The capacity of tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL) to trigger apoptosis preferentially in cancer cells, although sparing normal cells, has motivated clinical development of TRAIL receptor agonists as anti‐cancer therapeutics. The molecular mechanisms responsible for the differential TRAIL sensitivity of normal and cancer cells are, however, poorly understood. Here, we show a novel signalling pathway that activates cytoprotective autophagy in untransformed human epithelial cells treated with TRAIL. TRAIL‐induced autophagy is mediated by the AMP‐activated protein kinase (AMPK) that inhibits mammalian target of rapamycin complex 1, a potent inhibitor of autophagy. Interestingly, the TRAIL‐induced AMPK activation is refractory to the depletion of the two known AMPK‐activating kinases, LKB1 and Ca(2+)/calmodulin‐dependent kinase kinase‐β, but depends on transforming growth factor‐β‐activating kinase 1 (TAK1) and TAK1‐binding subunit 2. As TAK1 and AMPK are ubiquitously expressed kinases activated by numerous cytokines and developmental cues, these data are most likely to have broad implications for our understanding of cellular control of energy homoeostasis as well as the resistance of untransformed cells against TRAIL‐induced apoptosis.