Changes In Autophagy Levels Research Articles

Long-Term Administration of Nicotinamide Mononucleotide Mitigates High-Fat-Diet-Induced Physiological Decline in Aging Mice

BackgroundNicotinamide adenine dinucleotide (NAD+) levels decline with age, and boosting it can improve multi-organ functions and lifespan. ObjectivesNicotinamide mononucleotide (NMN) is a natural NAD+ precursor with the ability to enhance NAD+ biosynthesis. Numerous studies have shown that a high-fat diet (HFD) can accelerate the process of aging and many diseases. We hypothesized that long-term administration of NMN could exert protective effects on adipose, muscle, and kidney tissues in mice on an HFD act by affecting the autophagic pathway. MethodsMice at 14 mo of age were fed an HFD, and NMN was added to their drinking water at a dose of 400 mg/kg for 7 mo. The locomotor ability of the mice was assessed by behavioral experiments such as grip test, wire hang test, rotarod, and beam-walking test. At the end of the behavioral experiments, the pathological changes of each peripheral organ and the expression of autophagy-related proteins, as well as the markers of the senescence and inflammaging were analyzed by pathological staining, immunohistochemical staining, and western blotting, respectively. ResultsWe found that NMN supplementation increased NAD+ levels and ultimately attenuated age- and diet-related physiological decline in mice. NMN inhibited HFD-induced obesity, promoted physical activity, improved glucose and lipid metabolism, improved skeletal muscle function and renal damage, as well as mitigated the senescence and inflammaging as demonstrated by p16, interleukin 1β, and tumor necrosis factor α levels. In addition, the present study further emphasizes the potential mechanisms underlying the bidirectional relationship between NAD+ and autophagy. We detected changes in autophagy levels in various tissue organs, and NMN may play a protective role by inhibiting excessive autophagy induced by HFD. ConclusionsOur findings demonstrated that NMN administration attenuated HFD-induced metabolic disorders and physiological decline in aging mice.

New insights into the mechanisms and therapeutic strategies of chondrocyte autophagy in osteoarthritis.

Osteoarthritis (OA) is a chronic joint disease with an unclear cause characterized by secondary osteophytes and degenerative changes in the articular cartilage. More than 250 million people are expected to be affected by it by 2050, putting a tremendous socioeconomic strain on the entire world. OA cannot currently be treated with any effective medications that change the illness. Over time, chondrocytes undergo gradual metabolic, structural, and functional changes as a result of aging or abuse. The degenerative progression of osteoarthritis is significantly influenced by the imbalance of chondrocyte homeostasis. By continuously recycling and rebuilding macromolecules or organelles, autophagy functions as a crucial regulatory system to maintain homeostasis during an individual's growth and development. This review uses chondrocytes as its starting point and establishes a strong connection between autophagy and osteoarthritis in order to thoroughly examine the mechanisms behind chondrocyte autophagy in osteoarthritis. Biomarkers of chondrocyte autophagy will be identified, and prospective targeted medications and novel treatment approaches for slowing or preventing the course of OA will be developed based on chondrocyte senescence, autophagy, and apoptosis in OA. KEY MESSAGES: Currently, OA has not been treated with any drugs that can effectively cure it. We hope that by exploring specific targets in the course of osteoarthritis, we can promote the progress of treatment strategies. The degenerative progression of osteoarthritis is significantly influenced by the imbalance of chondrocyte balance. Through the continuous recovery and reconstruction of macromolecules or organelles, autophagy is an important regulatory system for maintaining homeostasis during individual growth and development. In this paper, the close relationship between autophagy and osteoarthritis was established with chondrocytes as the starting point, in order to further explore the mechanism of chondrocyte autophagy in osteoarthritis. The development process of osteoarthritis was studied from the perspective of chondrocytes, and the change of autophagy level had a significant impact on osteoarthritis. Chondrocyte autophagy is mainly determined by intracellular mitochondrial autophagy, so we are committed to finding relevant molecules. Through PI3K/AKT- and MAPK-related pathways, the biomarkers of chondrocyte autophagy were identified, and chondrocyte senescence, autophagy, and apoptosis based on osteoarthritis provided a constructive idea for the development of prospective targeted drugs and new therapies to slow down or prevent the progression of osteoarthritis.

Autophagy in erectile dysfunction: focusing on apoptosis and fibrosis.

In most types of erectile dysfunction, particularly in advanced stages, typical pathological features observed are reduced parenchymal cells coupled with increased tissue fibrosis. However, the current treatment methods have shown limited success in reversing these pathologic changes. Recent research has revealed that changes in autophagy levels, along with alterations in apoptosis and fibrosis-related proteins, are linked to the progression of erectile dysfunction, suggesting a significant association. Autophagy, known to significantly affect cell fate and tissue fibrosis, is currently being explored as a potential treatment modality for erectile dysfunction. However, these present studies are still in their nascent stage, and there are limited experimental data available. This review analyzes erectile dysfunction from a pathological perspective. It provides an in-depth overview of how autophagy is involved in the apoptotic processes of smooth muscle and endothelial cells and its role in the fibrotic processes occurring in the cavernosum. This study aimed to develop a theoretical framework for the potential effectiveness of autophagy in preventing and treating erectile dysfunction, thus encouraging further investigation among researchers in this area.

Curcumin regulates autophagy through SIRT3-SOD2-ROS signaling pathway to improve quadriceps femoris muscle atrophy in KOA rat model

Knee osteoarthritis (KOA) usually leads to quadriceps femoris atrophy, which in turn can further aggravate the progression of KOA. Curcumin (CUR) has anti-inflammatory and antioxidant effects and has been shown to be a protective agent for skeletal muscle. CUR has been shown to have a protective effect on skeletal muscle. However, there are no studies related to whether CUR improves KOA-induced quadriceps femoris muscle atrophy. We established a model of KOA in rats. Rats in the experimental group were fed CUR for 5 weeks. Changes in autophagy levels, reactive oxygen species (ROS) levels, and changes in the expression of the Sirutin3 (SIRT3)-superoxide dismutase 2 (SOD2) pathway were detected in the quadriceps femoris muscle of rats. KOA led to quadriceps femoris muscle atrophy, in which autophagy was induced and ROS levels were increased. CUR increased SIRT3 expression, decreased SOD2 acetylation and ROS levels, inhibited the over-activation of autophagy, thereby alleviating quadriceps femoris muscle atrophy and improving KOA. CUR has a protective effect against quadriceps femoris muscle atrophy, and KOA is alleviated after improvement of quadriceps femoris muscle atrophy, with the possible mechanism being the reduction of ROS-induced autophagy via the SIRT3-SOD2 pathway.

Knowledge domain and emerging trends of autophagy in cardiovascular research: A bibliometric analysis.

Autophagy is essential for the homeostasis and function of the cardiovascular system. Citespace is a visual analysis software developed in the context of scientometrics and data visualization. The purpose of this study is to use Citespace software to conduct bibliometric and visual analysis of the research on autophagy in cardiovascular diseases, identify the current status, hot spots and trends in this field, help researchers clarify the future research focus and direction of autophagy in cardiovascular diseases, and provide more positive and broader ideas for the treatment and drug development of cardiovascular diseases. In the Web of Science Core Collection database to download the data from 2004 to 2022 regarding autophagy in cardiovascular research. CitespaceV was used to collect the research status, hotspots and development trends for visual analysis. The 3568 articles were published by 547 authors from 397 institutions in 75 countries. From 2004 to 2021, the annual publications increased over time. The top 3 productive nations were China, the United States, and Germany. The leading institution was China's Fudan University. The most cited paper is Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). The research hotpots include monitoring methods for autophagy activity, changes in autophagy levels in different types of cardiovascular diseases, autophagy signal transduction mechanism in cardiovascular diseases, etc. Bibliometric analysis provided valuable information for autophagy research in cardiovascular disease, which is full of opportunities and challenges. The research of autophagy in the field of cardiovascular diseases is still worthy of in-depth exploration. A challenge with autophagy-targeted therapies is their dichotomy in which the goal is to target maladaptive autophagy while maintaining a baseline level of cell survival to optimize a beneficial outcome. It is necessary for scientists to develop new methods to evaluate the level of autophagy from basic application to human body and reveal the signaling mechanism of autophagy in different types of cardiovascular diseases.

Long non-coding RNAs: regulators of autophagy and potential biomarkers in therapy resistance and urological cancers.

The non-coding RNAs (ncRNAs) comprise a large part of human genome that mainly do not code for proteins. Although ncRNAs were first believed to be non-functional, the more investigations highlighted tthe possibility of ncRNAs in controlling vital biological processes. The length of long non-coding RNAs (lncRNAs) exceeds 200 nucleotidesand can be present in nucleus and cytoplasm. LncRNAs do not translate to proteins and they have been implicated in the regulation of tumorigenesis. On the other hand, One way cells die is by a process called autophagy, which breaks down proteins and other components in the cytoplasm., while the aberrant activation of autophagy allegedly involved in the pathogenesis of diseases. The autophagy exerts anti-cancer activity in pre-cancerous lesions, while it has oncogenic function in advanced stages of cancers. The current overview focuses on the connection between lncRNAs and autophagy in urological cancers is discussed. Notably, one possible role for lncRNAs is as diagnostic and prognostic variablesin urological cancers. The proliferation, metastasis, apoptosis and therapy response in prostate, bladder and renal cancers are regulated by lncRNAs. The changes in autophagy levels can also influence the apoptosis, proliferation and therapy response in urological tumors. Since lncRNAs have modulatory functions, they can affect autophagy mechanism to determine progression of urological cancers.

Impairment of Autophagy Mediates the Uric-Acid-Induced Phenotypic Transformation of Vascular Smooth Muscle Cells

Introduction: Hyperuricemia may be involved in the phenotypic transformation of vascular smooth muscle cells, thus promoting the occurrence of atherosclerosis, and autophagy may be one of the important links, but little is known about the specific molecular mechanism. Methods: We established a mouse model of hyperuricemia and studied the relationship between changes in autophagy levels and the phenotypic transformation of muscle cells. Results: Our study found that high uric acid levels promote the phenotypic transformation of muscle cells by inhibiting autophagy, thus enhancing their proliferation and migration abilities. If autophagy is restored, phenotypic transformation can be reversed by reducing the levels of the transcription factor Kruppel-like factor 4. Conclusion: Uric acid may induce the phenotypic transformation of muscle cells and promote the occurrence of atherosclerosis by disrupting normal autophagy.

Metformin attenuates inflammation and boosts autophagy in the liver and intestine of chronologically aged rats

BackgroundOur previous studies found that autophagy levels in liver and intestinal segments of naturally aging rats were downregulated, and the expression of pro-inflammatory factors was increased. The increased expression of pro-inflammatory factors might be related to the downregulation of autophagy. AMPK is the most critical upstream targeting and regulating molecule of autophagy, and Metformin, as an agonist of AMPK, has the effects of anti-inflammation and anti-aging. We pretreated 29-month-old naturally aging rats with Metformin for a short period and observed the changes in autophagy levels and pro-inflammatory factors in the liver, ileum, and colon after 31 days of intervention and preliminarily investigated the mechanism of its action. Methods29-month-old SPF male Wistar rats were divided into three groups: The control group, the Metformin 100 mg/kg intervention group, and the Metformin 250 mg/kg intervention group, with eight rats in each group. At 29 months, different concentrations of Metformin (100 mg/kg, 250 mg/kg) were given by gavage once a day until 30 months, and the control group was kept generally until 30 months. Western Blot was used to assess the expression levels of AMPK, P-AMPK, LC3, and P62 proteins in the liver and intestinal tissues. Intestinal and liver tissues were immunofluorescence labeled for LC3 and P62 proteins. Moreover, RT-qPCR was conducted to detect the expression levels of pro-inflammatory factors IL-1β, TNF-α, IL-6, and MMP-9 mRNA in liver and intestinal tissues. ResultsShort-term Metformin pretreatment (31 days) in naturally aging rats (29 months old) increased autophagy levels and down-regulated the expression of various pro-inflammatory cytokines (IL-1β, TNF-α, MMP-9, and IL-6) in various intestinal segments and the liver—the expression of LC3II protein enriched with the increase of Metformin concentration. The level of P62 protein decreased with the accumulation of Metformin concentration. And a higher concentration of Metformin was associated with increased expression of P-AMPK protein. ConclusionsMetformin intervention can boost the autophagy level in the liver and intestine and reduce the expression of aging-related inflammatory factors in aged rats, and these effects may be related to the increase of the AMPK phosphorylation level.

Autophagy increases the survival rate of Macrobrachium rosenbergiiin after Aeromonas hydrophila infection

Autophagy is known as crucial components of crustacean innate immunity against pathogens. The dynamic changes of autophagy level during infectious disease is apparently related to disease severity, which needs to deeply understand for a health development and management of aquaculture. In this study, the role of autophagy during A. hydrophila infection was elucidated in M. rosenbergii. Adult freshwater prawn was firstly administered A. hydrophila to assess the virulence of the isolated strain. Pathological analysis of hepatopancreas and intestine tissues of moribund prawn showed significantly pathological changes, including cellular swelling and vacuolation, contraction or karyorrhexis of nuclei, dissolution and necrosis of villi, structural destruction of the striate border. A significant apoptosis and autophagy was observed in hepatopancreas tissues of the infected shrimp. Moreover, the rapamycin-pretreated groups showed high survival rate in contrast with drastic high mortality in autophagy-suppressed groups. Further analysis showed that the elevated autophagy levels restricted apoptosis signals. We conclude that autophagy plays a positive role during A. hydrophila infection and may be is a new therapeutic target for bacterial disease.

Identification of Fangjihuangqi Decoction as a late-stage autophagy inhibitor with an adjuvant anti-tumor effect against non-small cell lung cancer

BackgroundClinically, although chemotherapy is one of the most commonly used methods of treating tumors, chemotherapeutic drugs can induce autophagic flux and increase tumor cell resistance, leading to drug tolerance. Therefore, theoretically, inhibiting autophagy may improve the efficacy of chemotherapy. The discovery of autophagy regulators and their potential application as adjuvant anti-cancer drugs is of substantial importance. In this study, we clarified that Fangjihuangqi Decoction (FJHQ, traditional Chinese medicine) is an autophagy inhibitor, which can synergistically enhance the effect of cisplatin and paclitaxel on non-small cell lung cancer (NSCLC) cells.MethodsWe observed the changes of autophagy level in NSCLC cells under the effect of FJHQ, and verified the level of the autophagy marker protein and cathepsin. Apoptosis was detected after the combination of FJHQ with cisplatin or paclitaxel, and NAC (ROS scavenger) was further used to verify the activation of ROS-MAPK pathway by FJHQ.ResultsWe observed that FJHQ induced autophagosomes in NSCLC cells and increased the levels of P62 and LC3-II protein expression in a concentration- and time-gradient-dependent manner, indicating that autophagic flux was inhibited. Co-localization experiments further showed that while FJHQ did not inhibit autophagosome and lysosome fusion, it affected the maturation of cathepsin and thus inhibited the autophagic pathway. Finally, we found that the combination of FJHQ with cisplatin or paclitaxel increased the apoptosis rate of NSCLC cells, due to increased ROS accumulation and further activation of the ROS-MAPK pathway. This synergistic effect could be reversed by NAC.ConclusionCollectively, these results demonstrate that FJHQ is a novel late-stage autophagy inhibitor that can amplify the anti-tumor effect of cisplatin and paclitaxel against NSCLC cells.

Resveratrol Ameliorates Fibrosis in Rheumatoid Arthritis-Associated Interstitial Lung Disease via the Autophagy–Lysosome Pathway

Interstitial lung disease associated with rheumatoid arthritis (RA-ILD) can lead to interstitial fibrosis and even lung failure as a complication of rheumatoid arthritis (RA), and there is currently no effective treatment and related basic research. Studies have found that resveratrol (Res) can improve the progression of RA by regulating autophagy, and increasing evidence supports the connection between autophagy and common interstitial lung disease (ILD). We explored changes in autophagy levels in fibrotic lungs in RA-ILD and found that the level of autophagy is enhanced in the early stage but inhibited in the late stage. However, resveratrol treatment improved the level of autophagy and reversed the inhibition of autophagy, and attenuated fibrosis. We created corresponding cell models that exhibited the same phenotypic changes as animal models; under the effect of resveratrol, the level of fibrosis changed accordingly, and the fusion process of lysosomes and autophagosomes in autophagy was liberated from the inhibition state. Resveratrol effects were reversed by the addition of the late autophagy inhibitor chloroquine. These results suggest that resveratrol attenuates pulmonary fibrosis, increases autophagic flux, and modulates the autophagy-lysosome pathway, and particularly it may work by improving the formation of autophagic lysosomes, which may be an effective treatment for induced RA-ILD.

Down-regulation of EVA1A by miR-103a-3p promotes hepatocellular carcinoma cells proliferation and migration

BackgroundEVA1A (Eva-1 homolog A), a novel protein involved in autophagy and apoptosis, functions as a tumor suppressor in some human primary cancers, including hepatocellular carcinoma (HCC). While it is consistently downregulated in several cancers, its involvement in hepatocarcinogenesis is still largely unknown.MethodsWe first detected the expression of EVA1A in HCC tissues and cell lines using RT‒qPCR, immunohistochemistry and western blotting and detected the expression of miR-103a-3p by RT‒qPCR. Then, bioinformatics prediction, dual-luciferase reporter gene assays and western blotting were used to screen and identify the upstream microRNA of EVA1A. After manipulating the expression of miR-103a-3p or EVA1A, wound healing, invasion, proliferation, colony formation, apoptosis, autophagy, mitosis and mitochondrial function assays, including mitochondrial membrane potential, ROS and ATP production assays, were performed to investigate the functions of miR-103a-3p targeting EVA1A in HCC cells. Apoptosis-related proteins were assessed by RT‒qPCR (TP53) or western blotting (TP53, BAX, Bcl-2 and caspase-3). Autophagy level was evaluated by observing LC3 puncta and examining the protein levels of p62, Beclin1 and LC3-II/I.ResultsWe found that EVA1A expression was decreased while miR-103a-3p expression was increased in HCC tissues and cell lines and that their expression was inversely correlated in HCC patients. The expression of miR-103a-3p was associated with HCC tumor stage and poor prognosis. miR-103a-3p could target EVA1A through direct binding to its 3'-UTR and suppress its expression. Overexpression of miR-103a-3p significantly downregulated the expression of EVA1A, TP53 and BAX, upregulated the JAK2/STAT3 pathway and promoted HCC cell migration, invasion and proliferation, while repression of miR-103a-3p dramatically upregulated the expression of EVA1A, TP53, BAX and cleaved-caspase-3, inhibited HCC cell migration, invasion and proliferation, and caused mitochondrial dysfunction and apoptosis. Overexpression of EVA1A significantly attenuated the cancer-promoting effects of miR-103a-3p in HCC cells, while knockdown of EVA1A alleviated the mitochondrial dysfunction and apoptosis caused by miR-103a-3p inhibition. Overexpression of EVA1A did not induce significant changes in autophagy levels, nor did it affect G2/M transition or mitosis.ConclusionThese findings indicate that the downregulation of the tumor suppressor EVA1A by miR-103a-3p potentially acts as a key mediator in HCC progression, mainly by inhibiting apoptosis and promoting metastasis. The miR-103a/EVA1A/TP53 axis provides a new potential diagnostic and therapeutic target for HCC treatment.

Low-energy green light alleviates senescence-like phenotypes in a cell model of photoaging.

Ultraviolet B (UVB) affects diverse pathways in skin cells, resulting in skin photoaging. Skin fibroblasts internalize and degrade elastin and collagen, playing prominent roles in photoaging. Green light is used in many fields of dermatology, but few studies have examined its role in photoaging. The present work aimed to assess low-energy green light for its effects in a previously proposed cell model of photoaging and to explore the possible anti-photoaging mechanism. The stress-induced premature senescence (SIPS) model was constructed via repeated treatment of MDFs with UVB. Senescence-like phenotypes were compared among normal, low-energy green light pretreatment and UVB groups, for example, cell morphological properties, senescence-associated β-galactosidase (SA-β-gal) amounts, extracellular matrix (ECM) biosynthesis and degradation, and autophagy. In comparison with the UVB group, the green light pretreatment group showed significantly decreased number of senescent mast cells and markedly declined signal intensity and amounts of SA-β-gal-positive cells. Furthermore, green light pretreatment directly affected ECM by increasing type I and type III collagen production and decreasing MMP-1 amounts. Moreover, changes in autophagy levels induced by green light pretreatment provided a potential mechanism underlying its anti-aging property. Low-energy green light pretreatment improves senescence-like phenotypes in vitro, indicating a possible application for anti-aging in clinic after future research has uncovered the potential mechanism.

P736: BCOR MUTATION REGULATES MYELODYSPLASTIC SYNDROMES PROGRESSION THROUGH REPRESSING AUTOPHAGY FLUX MEDIATED BY HDAC6 ACTIVATION

Background: Past studies have revealed that mutations in bcor are involved in various human cancers including MDS and AML. Autophagy plays a critical role in cancer progression. However, the role of bcor mutations in autophagy and MDS progression remains subject to debate. Aims: This study investigated the role of bcor mutations in autophagy and MDS progression. Methods: Cell lines with bcorp483L stable overexpression were established with a recombinant lentiviral vector. The effects of bcor mutations on cell proliferation, cell cycle progression, and cell apoptosis were detected by CCK8 assay, colony formation assay, and flow cytometry. We detected changes in autophagy levels by Western blot (WB), quantitative polymerase chain reaction (qPCR), and most importantly, transmission electron microscopy. The function of histone deacetylase 6 (HDAC6) was inhibited by Tubasatin A （TBA）, and We detected the consequent changes in the functional phenotype of the cells by WB and qPCR. The interactions between related proteins were analyzed by a co-immunoprecipitation assay. To test cell lines for mitochondrial dysfunction, bcorp483L mutant cells, negative control cells, and wild-type cells were double-stained with MitoTracker Green and MitoTracker Red, or single-stained with MitoSOX followed by fluorescence-activated cell sorting (FACS) analysis. The changes in pyroptosis level caused by bcorp483L mutation were detected by WB and qPCR. Results: The cell proliferative ability of bcorp483L mutant cells was repressed compared to the negative control cells. Besides, bcorp483L mutation could promote cell apoptosis and block the cell cycle progression to the G2 phase. Notably, bcorp483L mutation repressed autophagic flux in cultured cells, as corroborated by decreased expression of autophagy marker genes (e.g., ATG7, ATG10) and reduced numbers of autophagosomes or autolysosomes, which were accompanied by upregulation of HDAC6, downregulation of FoxO1 and ac-FOXO1. HDAC6 was required for inhibiting the deacetylation of FoxO1. Enhanced HDAC6 expression and repression of FOXO1 were required for bcorp483L -mutation-deduced autophagy. Depression of HDAC6 by TBA was sufficient for increasing the expression of FOXO1, ac-FOXO1, and inducing autophagic flux. Notably, we examined the direct interaction between HDAC6 and FOXO1, FOXO1 and ATG7. Bcorp483L mutated cells accumulated dysfunctional, non-respiring mitochondria and increased mitochondrial ROS production compared to negative control cells. And we found upward adjustment of pyroptosis level and DNA damage. Image:Summary/Conclusion:Bcor p.P483L mutation could disrupt BCOR protein structure and function, therefore reducing the transcriptional repressive effect of BCOR on HDAC6. HDAC6 was upregulated and decreases the acetylation of FOXO1, further repressing autophagy. We further verified the protein: protein interaction between HDAC6 and FOXO1, FOXO1 and ATG7. Defects in the clearance of damaged mitochondria by bcorp483L-mutation-regulated autophagy contribute to the accumulation of cellular reactive oxygen species（ROS）, thus promoting cell apoptosis and repressing cell proliferation. The escalation in pyroptosis levels was found after bcor mutations and may be partly due to increased ROS levels. We also found increased gene damage and increased likelihood of gene mutations in bcorp483L mutant cells, which would promote MDS transformation into AML. These findings indicate a completely novel BCOR-HDAC6-FOXO1-ATG7 axis in the regulation of autophagy and MDS progression, providing potential possibilities for MDS treatment.

4,4’-dimethoxychalcone increases resistance of mouse oocytes to postovulatory aging in vitro

Research questionDoes 4,4’-dimethoxychalcone (DMC), a natural antioxidant compound, effectively improve the quality of postovulatory ageing (POA) oocytes? DesignFreshly ovulated MII oocytes were cultured in 10-µM DMC for 12 h in vitro. Reactive oxygen species (ROS) production, apoptosis rate, mitochondrial distribution, formation of the actin cap, and fertilization and development potential of POA oocytes, were studied. The change of autophagy level was detected, and the autophagy inhibitor 3-methyladenine (3-MA) was used to establish the relationship between DMC and autophagy. ResultsDMC supplementation eliminated the accumulated ROS (P < 0.0001) and ROS dependent 4 hydroxynonenal products (P = 0.0399), and decreased apoptosis (P = 0.0033), reduced abnormal mitochondrion distribution (P = 0.0280), improved mitochondrial membrane potential (P = 0.0135) and restored the formation of the actin cap (P = 0.0487), thus improving fertilization ability (P = 0.0156) and developmental potential (P = 0.0130) in POA oocytes. The role autophagy plays in the effects of DMC supplementation was investigated. The immunofluorescence results showed that POA leads to the accumulation of SQSTM1/p62 (P = 0.0083) but DMC supplementation could eliminate this (P < 0.0001). The western blot result of p62 protein was similar to the immunofluorescence results of the POA group (P = 0.0441) and DMC supplementation group (P = 0.0154). After inhibiting autophagy by 3-MA, the DMC supplementation group could no longer eliminate the accumulation of ROS (P = 0.1704). ConclusionsDMC supplementation activates autophagy to protect oocytes from postovulatory ageing. This approach can feasibly improve the reproductive outcome of ART.

Dysregulation of autophagy-associated microRNAs in condyloma acuminatum.

Condyloma acuminatum, which is caused by low-risk human papillomavirus (lrHPV) infection, is one of the most common sexually transmitted diseases. Autophagy is thought to be associated with condyloma acuminatum, but how the autophagy process is regulated remains unclear. MicroRNAs (miRNAs) are important regulators of gene transcription that play a central role in many biological processes, including autophagy and viral infection. This study was designed to identify autophagy-related miRNAs and their targets in condyloma acuminatum and to validate their expression. The levels of the autophagy proteins microtubule-associated protein 1 light chain 3 (LC3) and P62/SQSTM1 (P62) were abnormally increased in the local lesion tissue of condyloma acuminatum patients compared with healthy controls. MiRNAs and their target mRNAs in condyloma acuminatum patients were analyzed by bioinformatics. Eighty-one differentially expressed miRNAs were identified, of which 56 were downregulated and 25 were upregulated. Two of the differentially expressed miRNAs associated with autophagy, miRNA-30a-5p and miRNA-514a-3p, were analyzed further, and their target genes were identified as autophagy-related protein (Atg) 5 and Atg12 and Atg3 and Atg12, respectively. The expression levels of miRNA-30a-5p and miRNA-514a-3p were decreased and those of Atg5, Atg12 and Atg3 were increased in condyloma acuminatum patients compared with healthy controls. In addition, miRNA-30a-5p and miRNA-514a-3p expression correlated with the proliferation index Ki-67 in condyloma acuminatum. Taken together, our results suggest that the changes in autophagy levels in patients with condyloma acuminatum may be related to the changes in miRNA-30a-5p and miRNA-514a-3p expression. This study provides a theoretical basis for identifying new mechanisms that link miRNAs, HPV infection and host autophagy in vivo.

Abstract 1063: Effectiveness of Tumor Treating Fields (TTFields) in combination with sorafenib for treatment of hepatocellular carcinoma in vitro and in vivo

Abstract Purpose/Objective(s): Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and it is one of the leading causes of related mortality worldwide. Sorafenib is an oral multikinase inhibitor that targets the Raf/MEK/ERK signaling pathway, thus inducing autophagy and blocking angiogenesis. Sorafenib is approved for advanced HCC and is the main first-line chemotherapy, yet its survival benefits are limited. Tumor Treating Fields (TTFields) therapy is an anticancer treatment that is non-invasively and locoregionally delivered to tumor bed via low intensity (1-3 V/cm), intermediate frequency (100-500 kHz), alternating electric fields. Since HCC is a complex, heterogeneous tumor with exhibited aberrant signaling pathways, sorafenib combined with additional chemotherapy agents and other types of treatment modalities, such as TTFields, may be a feasible option for targeting HCC. The purpose of this study was to explore the use of TTFields, alone and in combination with sorafenib, for HCC treatment. Materials/Methods: HCC cell lines (HepG2 and Huh-7D12) were treated for 72 hours with TTFields at various frequencies. Efficacy of TTFields and sorafenib combination was tested by applying optimal frequency TTFields in the presence of various concentrations of sorafenib. Cytotoxicity, apoptosis, and clonogenicity were determined, and overall effect was calculated as the product of the cytotoxic and clonogenic effects. Changes in autophagy levels were also examined. In vivo, N1S1 HCC cells (50,000) were orthotopically injected into the left hepatic lobe of SD rats. After 1 week, TTFields at the optimal frequency were continuously applied for 6 days to the abdominal region of rat torsos, and sorafenib (10 mg/kg/day) was injected daily. Tumor volume growth was determined by MRI. Results: The TTFields frequency assessed for optimally treating HCC cell lines was 150 kHz. Cells were sensitive to sorafenib in a dose-dependent manner, and concomitant addition of TTFields augmented this effect. The effect of TTFields, like that of sorafenib, was demonstrated to be related to increases in autophagic flux. In the animal model, tumor growth was significantly reduced in the combination group compared to other treatment groups. Conclusion: These results demonstrate that TTFields are effective for the treatment of HCC and may further enhance effectiveness in combination with standard of care chemotherapy. The ongoing phase 2 HEPANOVA (NCT03606590) clinical trial will investigate the safety and efficacy of TTFields plus sorafenib combination in patients with unresectable, locally advanced HCC. Citation Format: Shiri Davidi, Anna Shteingauz, Sara Jacobovitch, Karnit Gotlib, Catherine Tempel-Brami, Mijal Munster, Einav Zeevi, Eyal Dor-On, Rosa S. Schneiderman, Tali Voloshin, Adi Haber, Moshe Giladi, Adrian Kinzel, Uri Weinberg, Yoram Palti. Effectiveness of Tumor Treating Fields (TTFields) in combination with sorafenib for treatment of hepatocellular carcinoma in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1063.

Exercise Preconditioning Increases Beclin1 and Induces Autophagy to Promote Early Myocardial Protection via Intermittent Myocardial Ischemia-Hypoxia.

Exercise preconditioning (EP) provides protective effects for acute cardiovascular stress; however, its mechanisms need to be further investigated. Autophagy is a degradation pathway essential for myocardium health. Therefore, we investigated whether intermittent myocardial ischemia-hypoxia affected Beclin1 and whether the changes in autophagy levels contribute to EP-induced early myocardial protective effects. Rats were trained on a treadmill using an EP model (four cycles of 10 minutes of running/10 minutes of rest). Exhaustive exercise (EE) was performed to induce myocardial injury. Cardiac troponin I (cTnI) and ischemia-hypoxia staining were used to evaluate myocardial injury and protection. Double-labeled immunofluorescence staining and western blot analysis were employed to examine related markers. EP attenuated the myocardial ischemic-hypoxic injury induced by EE. Compared with the control (C) group, the dissociations of Beclin1/Bcl-2 ratio and Beclin1 expression were both higher in all other groups. Compared with the C group, PI3KC3 and the LC3-II/LC3-I ratio were higher in all other groups, whereas LC3-II was higher in the EE and EEP + EE groups. p62 was higher in the EE group than in the C group but lower in the EEP + EE group than in the EE group. We concluded that EP increases Beclin1 via intermittent myocardial ischemia-hypoxia and induces autophagy, which exerts early myocardial protective effects and reduces the myocardial ischemic-hypoxic injury induced by exhaustive exercise.

The imbalance of PGD2-DPs pathway is involved in the type 2 diabetes brain injury by regulating autophagy.

Prostaglandin D2 (PGD2) is the most abundant prostaglandin in the brain, but its involvement in brain damage caused by type 2 diabetes (T2D) has not been reported. In the present study, we found that increased PGD2 content is related to the inhibition of autophagy, which aggravates brain damage in T2D, and may be involved in the imbalanced expression of the corresponding PGD2 receptors DP1 and DP2. We demonstrated that DP2 inhibited autophagy and promotedT2D-induced brain damage by activating the PI3K/AKT/mTOR pathway, whereas DP1enhanced autophagy and amelioratedT2D brain damage by activating the cAMP/PKA pathway. In a T2D rat model, DP1 expression was decreased, and DP2 expression was increased; therefore, the imbalance in PGD2-DPs may be involved in T2D brain damage through the regulation of autophagy. However, there have been no reports on whether PKA can directly inhibit mTOR. The PKA catalytic subunit (PKA-C) has three subtypes (α, β and γ), and γ is not expressed in the brain. Subsequently, we suggested that PKA could directly interact with mTOR through PKA-C(α) and PKA-C(β). Our results suggest that the imbalance in PGD2-DPs is related to changes in autophagy levels in T2D brain damage, and PGD2 is involved in T2D brain damage by promoting autophagy via DP1-PKA/mTOR and inhibiting autophagy via DP2-PI3K/AKT/mTOR.

A study of the effects of SGLT-2 inhibitors on diabetic cardiomyopathy through miR-30d/KLF9/VEGFA pathway.

The aim of this study was to observe the protective effects of SGLT-2 inhibitors on diabetic cardiomyopathy. After constructing a diabetic rat model, the effects of SGLT-2 (Sodium-Glucose Cotransporter-2) inhibitors and miR-30d on cardiac function in rats were investigated by cardiac echocardiography, hematoxylin-eosin (HE) staining, and immunohistochemical methods. At the same time, changes in autophagy levels in rats were detected by Western blot (WB) experiments. SGLT-2 inhibitors improved the cardiac function of diabetic rats, and alleviated the pathological damage of myocardial tissue. Besides, knocking down miR-30d prevented the decrease of cardiac function in diabetic rats. Moreover, miR-30d could regulate the expression of the KLF9/VEGFA pathway and inhibit autophagy in rats. SGLT-2 inhibitors can regulate the autophagy level in diabetic rats through the miR-30d/KLF9/VEGFA pathway, thereby improving cardiac function.