Autophagic Levels Research Articles

Synergizing autophagic cell death and oxaliplatin-induced immunogenic death by a self-delivery micelle for enhanced tumor immunotherapy

Chemotherapy has become an emerging strategy to activate cytotoxic T cell responses by inducing immunogenic cell death (ICD), but the level of antitumor immunity induced by chemotherapeutic agents, such as oxaliplatin (OXA), is limited due to inadequate tumor antigen presentation and T cell activation. Inducing autophagic cell death (ACD) promotes the release of tumor antigen and the recruitment of dendritic cells, therefore strengthening antitumor immune responses. Here we simultaneously activate ICD and ACD with tumor targeting micelle to achieve enhanced antitumor chemo-immunotherapy. A self-delivery micelle is formulated by conjugating OXA prodrug with tocopherol succinate (TOS) as a hydrophobic segment and further encapsulates autophagy activator SMER28 to afford TOPR/SMER28, which specifically targets αvβ3 on tumor cells with c(RGDfK). Upon cellular internalization, OXA is released from the prodrug in response to the high concentration of reduced glutathione (GSH) in tumor cells, triggering ICD and releasing associated molecular patterns (DAMPs) signaling molecules to stimulate immunity. Meanwhile, SMER28 over-activates autophagy to induce autophagic cell death, which further leads to the maturation of dendritic cells and ultimately activates anti-tumor immune response. In the 4T1 tumor-bearing mice, the combination of OXA and SMER28 effectively inhibits tumor growth and activates antitumor immune responses. The tumor targeted micelle releases OXA and SMER28 in an on-demand profile and strengthens tumor chemo-immunotherapy by synergizing ICD and ACD, providing an alternative for antitumor immunotherapy. Statement of significanceChemotherapy induces immunogenic cell death (ICD) to activate anti-tumor immunity. However, the efficacy is limited by low levels of antigen presentation and T cell activation. To strengthen the antitumor immune responses induced by ICD, we first combine autophagic cell death (ACD) with ICD by formulating a glutathione-responsive oxaliplatin prodrug micelle co-encapsulating the autophagy activator SMER28. The activated autophagic level by SMER28 enhances the release of antigen and the recruitment of APCs, and ultimately bolsters T cell-mediated antitumor immune responses. We provide a potential strategy to amplify antitumor immune effects by combining autophagy activation with chemotherapy.

Adipose stem cell exosomes promote mitochondrial autophagy through the PI3K/AKT/mTOR pathway to alleviate keloids

BackgroundFibrosis with unrelieved chronic inflammation is an important pathological change in keloids. Mitochondrial autophagy plays a crucial role in reducing inflammation and inhibiting fibrosis. Adipose stem cell-derived exosomes, a product of adipose stem cell paracrine secretion, have pharmacological effects, such as anti-inflammatory and antiapoptotic effects, and mediate autophagy. Therefore, this study aims to investigate the function and mechanism of adipose stem cell exosomes in the treatment of keloids.MethodWe isolated adipose stem cell exosomes under normoxic and hypoxic condition to detect their effects on keloid fibroblast proliferation, migration, and collagen synthesis. Meanwhile, 740YPDGFR (PI3K/AKT activator) was applied to detect the changes in autophagic flow levels and mitochondrial morphology and function in keloid fibroblasts. We constructed a human keloid mouse model by transplanting human keloid tissues into six-week-old (20–22 g; female) BALB/c nude mice, meanwhile, we applied adipose stem cell exosomes to treat the mouse model and observed the retention and effect of ADSC exosomes in vivo.ResultsADSC exosomes can inhibit the PI3K/AKT/mTOR signaling pathway. The exosomes of ADSCs decreased the inflammatory level of KFs, enhanced the interaction between P62 and LC3, and restored the mitochondrial membrane potential. In the human keloid mouse model, ADSC exosomes can exist stably, promote mitochondrial autophagy in keloid tissue, improve mitochondrial morphology, reduce inflammatory reaction and fibrosis. Meanwhile, At the same time, the exosomes derived from hypoxic adipose stem cells have played a more effective role in both in vitro and in vivo experiments.ConclusionsAdipose stem cell exosomes inhibited the PI3K/AKT/mTOR pathway, activated mitochondrial autophagy, and alleviated keloid scars.Graphical

AoRan1 Is Involved in Regulating Conidiation, Stress Resistance, Secondary Metabolism, and Pathogenicity in Arthrobotrys oligospora.

Arthrobotrys oligospora is a representative nematode-trapping (NT) fungus that is able to capture, kill, and digest nematodes by producing specialized three-dimensional networks (traps) under nutrient-deprived conditions. Ran1 is a serine/threonine protein kinase that can act as a negative regulator of sexual conjugation and meiosis. However, the specific role of Ran1 remains largely unknown in NT fungi. Here, we identified AoRan1 (AOL_s00004g277) via gene disruption, phenotypic analysis, and metabolomic analysis. Our findings reveal that Aoran1 knockout caused a remarkable increase in conidial production, traps, and nematode feeding efficiency. In addition, the absence of Aoran1 resulted in the accumulation of lipid droplets and increased autophagic levels as well as increased tolerance to cell wall synthesis-disturbing reagents and oxidants. Metabolomic analyses also suggested that AoRan1 is involved in multiple metabolic processes, such as fatty acid biosynthesis. In summary, our results suggest that AoRan1 is crucial in conidiation, pathogenicity, and secondary metabolism. This study's results further our understanding of the molecular mechanisms by which AoRan1 regulates conidiation and trap formation in A. oligospora.

Autophagic genes and antioxidant status during doxorubicin therapy under temperature-stressed conditions in breast cancer cells

ObjectivesAutophagy as a cellular event swings between apoptosis induction and cellular maintenance during chemotherapy. The interplay between autophagy and reactive oxygen species (ROS) remains obscure in cancer progression and treatment. This study aimed to determine the combination effect of chemotherapy and hyperthermia on cancer cell proliferation, autophagy signaling, and oxidative stress status. MethodsTo estimate the autophagic genes' role and antioxidant capacity involvement during chemotherapy, human breast cancer cell lines (MCF7 and CAL51) were exposed to a high temperature at 42 °C and treated with doxorubicin (DOX). The MTT assays were performed to determine the cell survival levels, and the autophagic gene expression levels (ATG5, LC3A, LC3B, and Beclin1) were analyzed. The intracellular TAS (total antioxidant status) and SOD (superoxide dismutase) activities were determined. ResultsThe combined effects of high temperature with chemotherapy significantly reduced cell viability and cell survival compared to cells treated with the chemotherapeutic agent DOX alone. The cell's autophagic gene activities were significantly increased after exposure to 42 °C temperature and DOX-based chemotherapy compared to the cells treated with DOX alone. It was observed that TAS and SOD activities were increased in the cell lines exposed to high temperatures with DOX compared to the cells treated with DOX alone. Increased autophagic gene expression levels and cell death were observed in response to the high temperature and DOX treatment in breast cancer cells. ConclusionThe study may provide a plausible route and treatment plan for the individual adoption of cancer chemotherapy and be developed as part of the personalized medicine protocol.

Saikosaponin D Inhibits Lung Metastasis of Colorectal Cancer Cells by Inducing Autophagy and Apoptosis.

Saikosaponin D (SSD), derived from Bupleurum falcatum L., has various pharmacological properties, including immunoregulatory, anti-inflammatory, and anti-allergic effects. Several studies have investigated the anti-tumor effects of SSD on cancer in multiple organs. However, its role in colorectal cancer (CRC) remains unclear. Therefore, this study aimed to elucidate the suppressive effects of SSD on CRC cell survival and metastasis. SSD reduced the survival and colony formation ability of CRC cells. SSD-induced autophagy and apoptosis in CRC cells were measured using flow cytometry. SSD treatment increased LC3B and p62 autophagic factor levels in CRC cells. Moreover, SSD-induced apoptosis occurred through the cleavage of caspase-9, caspase-3, and PARP, along with the downregulation of the Bcl-2 family. In the in vivo experiment, a reduction in the number of metastatic tumor nodules in the lungs was observed after the oral administration of SSD. Based on these results, SSD inhibits the metastasis of CRC cells to the lungs by inducing autophagy and apoptosis. In conclusion, SSD suppressed the proliferation and metastasis of CRC cells, suggesting its potential as a novel substance for the metastatic CRC treatment.

Targeting the autophagy-NAD axis protects against cell death in Niemann-Pick type C1 disease models

Impairment of autophagy leads to an accumulation of misfolded proteins and damaged organelles and has been implicated in plethora of human diseases. Loss of autophagy in actively respiring cells has also been shown to trigger metabolic collapse mediated by the depletion of nicotinamide adenine dinucleotide (NAD) pools, resulting in cell death. Here we found that the deficit in the autophagy-NAD axis underpins the loss of viability in cell models of a neurodegenerative lysosomal storage disorder, Niemann-Pick type C1 (NPC1) disease. Defective autophagic flux in NPC1 cells resulted in mitochondrial dysfunction due to impairment of mitophagy, leading to the depletion of both the reduced and oxidised forms of NAD as identified via metabolic profiling. Consequently, exhaustion of the NAD pools triggered mitochondrial depolarisation and apoptotic cell death. Our chemical screening identified two FDA-approved drugs, celecoxib and memantine, as autophagy activators which effectively restored autophagic flux, NAD levels, and cell viability of NPC1 cells. Of biomedical relevance, either pharmacological rescue of the autophagy deficiency or NAD precursor supplementation restored NAD levels and improved the viability of NPC1 patient fibroblasts and induced pluripotent stem cell (iPSC)-derived cortical neurons. Together, our findings identify the autophagy-NAD axis as a mechanism of cell death and a target for therapeutic interventions in NPC1 disease, with a potential relevance to other neurodegenerative disorders.

Neurokinin-1 receptor antagonist aprepitant regulates autophagy and apoptosis via ROS/JNK in intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA) has a poor prognosis and limited treatment options. Aprepitant, a selective NK-1R antagonist, can inhibit the growth of various tumours invitro and invivo. However, it remains unclear whether aprepitant has cytotoxic effects on iCCA. We measured the expression of SP/NK-1R in clinical samples of iCCA by immunohistochemistry. Then, we detected the cytotoxic effects of aprepitant on iCCA cells via MTT, EdU and colony formation assay. We constructed a subcutaneous xenograft model of BALB/c nude mice by using HCCC-9810 and RBE cell lines to explore the effects of aprepitant invivo. To elucidate the potential mechanisms, we explored the pro-apoptotic effect of aprepitant by flow cytometric, western blotting, ROS detection and JC-1 staining. Furthermore, we detected the autophagic level of HCCC-9810 and RBE by western blotting, mRFP-eGFP-LC3 adenovirus transfection and electron microscope. SP/NK-1R is significantly expressed in iCCA. Aprepitant inhibited human iCCA xenograft growth and dose-dependently decreased the viability of RBE and HCCC-9810 cells. Aprepitant-induced mitochondria-dependent apoptosis through ROS/JNK pathway. Additionally, pretreatment with z-VAD-fmk partly reversed the effect of aprepitant on cell viability, while NAC completely attenuated the cytotoxic effects of aprepitant invitro. Furthermore, we observed the dynamic changes of autophagosome in RBE and HCCC-9810 cells treated with aprepitant. SP/NK-1R signalling is significantly activated in iCCA and promotes the proliferation of iCCA cells. By contrast, aprepitant can induce autophagy and apoptosis in iCCA cells via ROS accumulation and subsequent activation of JNK.

Modulation of autophagy affected tumorigenesis induced by the envelope glycoprotein of JSRV

Ovine pulmonary adenocarcinoma (OPA), caused by the jaagsiekte sheep retrovirus (JSRV), is a chronic, progressive, and contagious lung tumor that seriously affects sheep production. It also represents a valuable animal model for several human lung adenocarcinomas. However, little is known about the role of autophagy in OPA tumorigenesis. Here, Western blotting combined with transmission electron microscopy examination and Cyto-ID dye staining was employed for evaluation of changes of autophagic levels. The results of the present study showed that expression of the autophagy marker proteins Beclin-1 and LC3 was decreased in OPA lung tissues, as well as in cells overexpressing the envelope glycoprotein of JSRV (JSRV Env). Reduced numbers of autophagosomes were also observed in cells overexpressing JSRV Env, although assessment of autophagic flux showed that JSRV Env overexpression did not block the formation of autophagosomes, suggesting increased degradation of autolysosomes. Last, mouse xenograft experiments indicated that inhibition of autophagy by 3-methyladenine suppressed both tumor growth and the epithelial-to-mesenchymal transition. In conclusion, JSRV, through JSRV Env, takes advantage of the autophagy process, leading to the development of OPA.

Pparα activation stimulates autophagic flux through lipid catabolism-independent route.

Autophagy is a cellular process that involves the fusion of autophagosomes and lysosomes to degrade damaged proteins or organelles. Triglycerides are hydrolyzed by autophagy, releasing fatty acids for energy through mitochondrial fatty acid oxidation (FAO). Inhibited mitochondrial FAO induces autophagy, establishing a crosstalk between lipid catabolism and autophagy. Peroxisome proliferator-activated receptor α (PPARα), a transcription factor, stimulates lipid catabolism genes, including fatty acid transport and mitochondrial FAO, while also inducing autophagy through transcriptional regulation of transcription factor EB (TFEB). Therefore, the study explores whether PPARα regulates autophagy through TFEB transcriptional control or mitochondrial FAO. In aquaculture, addressing liver lipid accumulation in fish is crucial. Investigating the link between lipid catabolism and autophagy is significant for devising lipid-lowering strategies and maintaining fish health. The present study investigated the impact of dietary fenofibrate and L-carnitine on autophagy by activating Pparα and enhancing FAO in Nile tilapia (Oreochromis niloticus), respectively. The dietary fenofibrate and L-carnitine reduced liver lipid content and enhanced ATP production, particularly fenofibrate. FAO enhancement by L-carnitine showed no changes in autophagic protein levels and autophagic flux. Moreover, fenofibrate-activated Pparα promoted the expression and nuclear translocation of Tfeb, upregulating autophagic initiation and lysosomal biogenesis genes. Pparα activation exhibited an increasing trend of LC3II protein at the basal autophagy and cumulative p62 protein trends after autophagy inhibition in zebrafish liver cells. These data show that Pparα activation-induced autophagic flux should be independent of lipid catabolism.

Nur77 increases mitophagy and decreases aggregation of α-synuclein by modulating the p-c-Abl/p-PHB2 Y121 in α-synuclein PFF SH-SY5Y cells and mice

Parkinson's disease (PD) is characterized by the progressive death of dopamine (DA) neurons and the pathological accumulation of α-synuclein (α-syn) fibrils. In our previous study, simulated PHB2 phosphorylation was utilized to clarify the regulatory role of c-Abl in PHB2-mediated mitophagy in PD models. In this investigation, we employed an independently patented PHB2Y121 phosphorylated antibody in the PD model to further verify that the c-Abl inhibitor STI571 can impede PHB2Y121 phosphorylation, decrease the formation of α-Syn polymers, and improve autophagic levels. The specific involvement of Nur77 in PD pathology has remained elusive. We also investigate the contribution of Nur77, a nuclear transcription factor, to α-syn and mitophagy in PD. Our findings demonstrate that under α-syn, Nur77 translocates from the cytoplasm to the mitochondria, improving PHB-mediated mitophagy by regulating c-Abl phosphorylation. Moreover, Nur77 overexpression alleviates the expression level of pS129-α-syn and the loss of DA neurons in α-syn PFF mice, potentially associated with the p-c-Abl/p-PHB2 Y121 axis. This study provides initial in vivo and in vitro evidence that Nur77 protects PD DA neurons by modulating the p-c-Abl/p-PHB2 Y121 axis, and STI571 holds promise as a treatment for PD.

2'-Hydroxychalcone Induces Autophagy and Apoptosis in Breast Cancer Cells via the Inhibition of the NF-κB Signaling Pathway: In Vitro and In Vivo Studies.

2'-Hydroxychalcone is a hydroxyl derivative of chalcones, which are biosynthetic precursors of flavonoids and rich in the human diet. The anticancer activity of 2'-hydroxychalcone has been reported in several cancers but remains to be investigated in breast cancer. In the current study, 2'-hydroxychalcone showed significant cytotoxicity against breast cancer cell lines MCF-7 and CMT-1211. It could inhibit breast cancer cell proliferation, migration, and invasion in vitro and suppress tumor growth and metastasis in vivo. Mechanistic investigation revealed that the NF-κB pathway was significantly inhibited by 2'-hydroxychalcone treatment accompanied by an excessive intracellular accumulation of reactive oxygen species, induction of endoplasmic reticulum stress, and activation of JNK/MAPK. In addition, 2'-hydroxychalcone elevated the autophagic levels in breast cancer cells equipped with increasing numbers of autophagy vesicles and complete autophagic flux. Finally, autophagy-dependent apoptosis was observed in 2'-hydroxychalcone-induced cell death. In conclusion, 2'-hydroxychalcone enhances the autophagic levels and induces apoptosis in breast cancer cells, which could be contributed to the inhibition of the pro-survival NF-κB signaling, indicating a promising potential for 2'-hydroxychalcone in future anticancer drug development.

Alpha-mangostin decreases high glucose-induced damage on human umbilical vein endothelial cells by increasing autophagic protein expression.

Diabetes is a chronic disorder that occurs as a result of impaired glucose metabolism. In hyperglycaemic states, the balance between oxidative stress and antioxidant enzymes is disrupted leading to oxidative damage and cell death. In addition, impaired autophagy leads to the storage of dysfunctional proteins and cellular organelles in the cell. Hence, the cytoprotective function of autophagy may be disrupted by high glucose conditions. Alpha-mangostin (A-MG) is an essential xanthone purified from the mangosteen fruit. The different pharmacological benefits of alpha-mangostin, including antioxidant, anti-obesity, and antidiabetic, were demonstrated. We evaluated the protective influence of A-MG on autophagic response impaired by high concentrations of glucose in human umbilical vein endothelial cells (HUVECs). The HUVECs were treated with various glucose concentrations (5-60 mM) and A-MG (1.25-10 μM) for three days. Then, HUVECs were treated with 60 mM of glucose+2.5 μM of A-MG to measure viability, ROS, and NO content. Finally, the levels of autophagic proteins including LC3, SIRT1, and beclin 1 were evaluated by western blot. The results expressed that high glucose condition (60 mM) decreased viability and increased ROS and NO content in HUVECs. In addition, LC3, SIRT1, and beclin 1 protein levels declined when HUVECs were exposed to the high concentration of glucose. A-MG reversed these detrimental effects and elevated autophagic protein levels. Our data represent that A-MG protects HUVECs against high glucose conditions by decreasing ROS and NO generation as well as increasing the expression of autophagy proteins.

Autophagy Contributes to Homeostasis in Esophageal Epithelium Where High Autophagic Vesicle Level Marks Basal Cells With Limited Proliferation and Enhanced Self-Renewal Potential

Background & AimsAutophagy plays roles in esophageal pathologies both benign and malignant. Here, we aim to define the role of autophagy in esophageal epithelial homeostasis. MethodsWe generated tamoxifen-inducible, squamous epithelial-specific Atg7 (autophagy related 7) conditional knockout mice to evaluate effects on esophageal homeostasis and response to the carcinogen 4-nitroquinoline 1-oxide (4NQO) using histological and biochemical analyses. We FACS sorted esophageal basal cells based upon fluorescence of the autophagic vesicle (AV)-identifying dye Cyto-ID, then subjected these cells to transmission electron microscopy, image flow cytometry, 3D organoid assays, RNA-Sequencing (RNA-Seq), and cell cycle analysis. 3D organoids were subjected to passaging, single cell (sc) RNA-Seq, cell cycle analysis, and immunostaining. ResultsGenetic autophagy inhibition in squamous epithelium resulted in increased proliferation of esophageal basal cells under homeostatic conditions and also was associated with significant weight loss in mice treated with 4NQO that further displayed perturbed epithelial tissue architecture. Esophageal basal cells with high AV level (Cyto-IDHigh) displayed limited organoid formation capability upon initial plating but passaged more efficiently than their counterparts with low AV level (Cyto-IDLow). RNA-Seq suggested increased autophagy in Cyto-IDHigh esophageal basal cells along with decreased cell cycle progression, the latter of which was confirmed by cell cycle analysis. scRNA-Seq of 3D organoids generated by Cyto-IDLow and Cyto-IDHigh cells identified expansion of 3 cell populations, and enrichment of G2/M-associated genes in the Cyto-IDHigh group. Ki67 expression was also increased in organoids generated by Cyto-IDHigh cells, including in basal cells localized beyond the outermost cell layer. ConclusionsAutophagy contributes to maintenance of the esophageal proliferation-differentiation gradient. Esophageal basal cells with high AV level exhibit limited proliferation and generate 3D organoids with enhanced self-renewal capacity.

Diminished LC3 expression with unchanged Beclin 1 levels in right atrial appendage tissue of diabetic patients undergoing coronary artery bypass graft

Type 2 diabetes potentiates the risk of heart failure. A vital physiologic process, autophagy, may be impaired in the diabetic heart. The purpose of the present work was to explore the autophagic status in the human diabetic heart. Techniques like immunohistochemistry and western blotting were employed to examine the expression of some of the important proteins involved in autophagic machinery. Our brief study reports, for the first time, evidence of decreased cardiac autophagic levels in diabetic patients.

Phenol-Soluble Modulin α3 Stimulates Autophagy in HaCaT Keratinocytes.

Phenol-soluble modulins (PSMs) are pore-forming toxins (PFTs) produced by staphylococci. PSMs exert diverse cellular effects, including lytic, pro-apoptotic, pro-inflammatory and antimicrobial actions. Since the effects of PSMs on autophagy have not yet been reported, we evaluated the autophagic activity in HaCaT keratinocytes treated with recombinant PSMα3. The autophagic flux and levels of autophagic marker proteins were determined using Western blot analysis. Subcellular localization of LC3B and Beclin-1 was investigated using an indirect immunofluorescence assay. The ultrastructural features of control and PSMα3-treated cells were evaluated via transmission electron microscopy. Cytoplasmic acidification was measured via acridine orange staining. Phosphorylation levels of protein kinases, implicated in autophagy regulation, were studied using a phospho-kinase array and Western blot analysis. PSMα3 facilitated the intracellular redistribution of LC3B, increased the average number of autophagosomes per cell, promoted the development of acidic vesicular organelles, elevated the levels of LC3B-II, stimulated autophagic flux and triggered a significant decrease in the net autophagic turnover rate. PSMα3 induced the accumulation of autophagosomes/autolysosomes, amphisomes and multilamellar bodies at the 0.5, 6 and 24 h time points, respectively. The phospho-Akt1/2/3 (T308 and S473), and phospho-mTOR (S2448) levels were decreased, whereas the phospho-Erk1/2 (T202/Y204 and T185/Y187) level was increased in PSMα3-treated cells. In HaCaT keratinocytes, PSMα3 stimulates autophagy. The increased autophagic activity elicited by sub-lytic PSM concentrations might be an integral part of the cellular defense mechanisms protecting skin homeostasis.

The Newly Proposed Mechanism of Cardiomyocyte Protection of Carvedilol- Anti-Apoptosis Pattern of Carvedilol in Anoxia by Inducing Autophagy Partly through the AMPK/mTOR Pathway

Purpose: To investigate the underlying mechanism of cardiomyocyte protection of carvedilol based on autophagy and apoptosis. Methods: Neonatal rat ventricular myocytes (NRVMs) were exposed to various concentrations of carvedilol before anoxia, and pretreated with 3-MA or compound C for inhibiting autophagy or p-AMPK expression. CCK-8 colorimeter and flow cytometry were used to determine the cell viability and apoptotic rates. The variation of mRNA and protein was measured by RT-PCR and Western blot. The presence of autophagosomes was observed by electron microscopy. Results: First, we found that carvedilol increased autophagic marker levels in a concentration-dependent manner and the number of autophagosomes in NRVMs. Moreover, carvedilol substantially enhanced the viability and noticeably reduced the CK, MDA and LDH levels and cell apoptosis rate compared with the anoxia group. In addition, carvedilol decreased the levels of caspase-3 and Bim in mRNA and protein, but such effect was blocked by the special autophagy inhibitor-3-MA, and the number of autophagosomes was significantly decreased when treated with 3-MA, indicating that carvedilol exhibited anti-apoptotic and anti-injury effects by inducing autophagy in anoxia NRVMs, but these effects can be abolished by adding 3-MA to suppress autophagy. Finally, the carvedilol treatment-induced autophagy by enhancing the activation of p-AMPK and inhibiting p-mTOR. Electron microscopy presented that the number of autophagosomes was significantly decreased when treating with compound C, indicating that carvedilol induced autophagy in anoxia NRVMs partly by the AMPK-mTOR signaling pathway. Conclusions: Carvedilol has cardioprotection by inducing autophagy against apoptosis partly through the AMPK/mTOR pathway during anoxia in NRVMs.

FGF2 Rescued Cisplatin-Injured Granulosa Cells through the NRF2-Autophagy Pathway.

Premature ovarian failure (POF) is a complicated disorder related to the apoptosis of granulosa cells. The incidence of chemotherapy-associated POF is rising dramatically owing to the increasing proportion of cancer in adolescents. According to previous studies, oxidative stress caused by chemotherapeutic agents plays an important role in the development of POF. However, the exact effects of nuclear factor-erythroid 2-related factor2 (NRF2), a pivotal anti-oxidative factor, are still unknown in chemotherapy-associated POF. Firstly, we manipulated NRF2 expressions on a genetic or pharmaceutical level in cisplatin-injured granulosa cell models. The results indicate that the increasing NRF2 in cisplatin-injured cells was just compensatory and not enough to resist the accumulated stress. Upregulation of NRF2 could protect granulosa cells against cisplatin via elevating autophagic level by using an autophagic activator (rapamycin) and inhibitor (chloroquine). Additionally, exogenous FGF2 exerted a protective role by increasing NRF2 expression and promoting its nuclear translocation. Meanwhile, the results in cisplatin-POF mice models were consistent with what was found in injured cells. In conclusion, our research proved that FGF2 rescued cisplatin-injured granulosa cells through the NRF2-autophagy pathway and might provide a possible alternative treatment choice by targeting NRF2 for POF patients who are intolerant or unsuitable to FGF2.

The miR-224-5p/SIRT3/AMPK/mTOR axis is involved in the melatonin-mediated inhibition of glucocorticoid-induced osteoporosis by activating autophagy.

Melatonin has been shown to exert an inhibitory effect on osteoporosis. This study investigates the function of the miR-224-5p/SIRT3/AMPK/mTOR axis in melatonin-mediated effects against osteoporosis. Human bone marrow mesenchymal stem cells (hBMSCs) were treated with glucocorticoid dexamethasone to induce an in vitro osteoporosis model. After melatonin treatment, miR-224-5p and SIRT3 levels were measured by RT‒PCR. Transmission electron microscopy and immunofluorescence were conducted for evaluating autophagy. Western blotting was carried out to determine the expression of osteogenesis-related proteins (Runx2, OSX, OPN, and OCN), SIRT3-AMPK-mTOR axis, and autophagy-related markers (LC3 and p62). Alizarin red staining was used to measure matrix mineralization. The data showed that melatonin inhibited dexamethasone-induced osteoporosis in vitro, and enhanced autophagic levels (as indicated by increased LC3 puncta, LC3II/I ratio, and autophagic vacuoles). In terms of the mechanisms, melatonin decreased miR-224-5p expression and increased SIRT3. SRIT3 was shown to be a direct target of miR-224-5p. miR-224-5p upregulation or SIRT3 downregulation reversed the effects of melatonin on osteoporosis and suppressed autophagy. Additionally, miR-224-5p inhibited SIRT3 expression and AMPK pathway activation. In summary, we discovered that melatonin suppressed glucocorticoid-induced osteoporosis and autophagy inhibition via the miR-224-5p/SIRT3/AMPK/mTOR axis.

Exogenous galanin alleviates hepatic steatosis by promoting autophagy via the AMPK-mTOR pathway

Defective autophagy-induced intracellular lipid degradation is causally associated with non-alcoholic fatty liver disease (NAFLD) development. Therefore, agents that can restore autophagy may have potential clinical application prospects on this public health issue. Galanin (GAL) is a pleiotropic peptide that regulates autophagy and is a potential drug for the treatment of NAFLD. In this study, we used an MCD-induced NAFLD mouse model in vivo and an FFA-induced HepG2 hepatocyte model in vitro to evaluate the anti-NAFLD effect of GAL. Exogenous GAL supplementation significantly attenuated lipid droplet accumulation and suppressed hepatocyte TG levels in mice and cell models. Mechanistically, Galanin-mediated reduction of lipid accumulation was positively correlated with upregulated p-AMPK, as evidenced by upregulated protein expressions of fatty acid oxidation-related gene markers (PPAR-α and CPT1A), upregulated expressions of the autophagy-related marker (LC3B), and downregulated autophagic substrate p62 levels. In FFA-treated HepG2 cells, activation of fatty acid oxidation and autophagy-related proteins by galanin was reversed by autophagy inhibitors, chloroquine, and the AMPK inhibitor. Galanin ameliorates hepatic fat accumulation by inducing autophagy and fatty acid oxidation via the AMPK/mTOR pathway.

Hepatic and Skeletal Muscle Autophagy Marker Levels in Rat Models of Prenatal and Postnatal Protein Restriction.

Fetal growth restriction (FGR) leads to adult-onset metabolic syndrome. Intrauterine and early postnatal caloric restriction ameliorates the risk in animal models. To understand the underlying mechanism, we compared autophagic marker levels between offspring with FGR and those with prenatal and early postnatal protein restriction (IPPR). We postulated that FGR would impair, whereas IPPR would help regulate, autophagy in neonatal rats. This study involved control (Con), FGR offspring (Pre), and IPPR offspring groups (Pre + Post); n = 5/group. We assessed the abundance of autophagy markers in the liver and skeletal muscles. At birth, the Pre group pups had lower levels of some autophagy-related proteins, with increased p62 expression and a low microtubule-associated protein light chain beta (LC3-II:LC3-I) ratio. This finding suggests a lower hepatic autophagy flux in FGR offspring than the Con group. The hepatic levels of autophagy proteins were considerably decreased in the Pre and Pre + Post groups at 21 days of age compared to the Con group, but the LC3-II:LC3-I ratio was higher in the Pre + Post group than in the Con and Pre groups. The muscle levels of beclin-1, LC3-II, and p62 were lower in the Pre group pups, with no difference in the LC3-II:LC3-I ratio among the groups. An imbalance in the nutritional environment is associated with downstream autophagic flux, thus suggesting that FGR offspring will have impaired autophagic flux, and that post-natal nutrition restriction might help reduce this risk.