Inhibition of anlotinib-induced autophagy attenuates invasion and migration by regulating epithelial-mesenchymal transition and cytoskeletal rearrangement through ATG5 in human osteosarcoma cells

Abstract The cure rates for osteosarcoma have remained unchanged in the past three decades, especially for patients with pulmonary metastasis. Thus, a new and effective treatment for metastatic osteosarcoma is urgently needed. Anlotinib has been reported to have antitumor effects on advanced osteosarcoma. However, both the effect of anlotinib on autophagy in osteosarcoma and the mechanism of anlotinib-mediated autophagy in pulmonary metastasis are unclear. The effect of anlotinib treatment on the metastasis of osteosarcoma was investigated by transwell assays, wound healing assays, and animal experiments. Related proteins were detected by western blotting after anlotinib treatment, ATG5 silencing, or ATG5 overexpression. Immunofluorescence staining and transmission electron microscopy were used to detect alterations in autophagy and the cytoskeleton. Anlotinib inhibited the migration and invasion of osteosarcoma cells but promoted autophagy and increased ATG5 expression. Furthermore, the decreases in invasion and migration induced by anlotinib treatment were enhanced by ATG5 silencing. In addition, Y-27632 inhibited cytoskeletal rearrangement, which was rescued by ATG5 overexpression. ATG5 overexpression enhanced epithelial-mesenchymal transition (EMT). Mechanistically, anlotinib-induced autophagy promoted migration and invasion by activating EMT and cytoskeletal rearrangement through ATG5 both in vitro and in vivo. Our results demonstrated that anlotinib can induce protective autophagy in osteosarcoma cells and that inhibition of anlotinib-induced autophagy enhanced the inhibitory effects of anlotinib on osteosarcoma metastasis. Thus, the therapeutic effect of anlotinib treatment can be improved by combination treatment with autophagy inhibitors, which provides a new direction for the treatment of metastatic osteosarcoma.

Atg-mediated autophagy,exercise and skeletal muscle aging / 中国组织工程研究

BACKGROUND:Exercise as a viable non-pharmacological treatment has the potential to reverse skeletal muscle aging that deteriorates with age.The role of autophagy in the skeletal muscle aging process is indispensable.During skeletal muscle aging,Atg genes involved in regulating autophagy regulate the autophagic process in either a facilitative or inhibitory manner to improve the physiological morphology of skeletal muscle.However the specific molecular mechanisms of autophagy in the exercise regulation of skeletal muscle aging remain puzzling. OBJECTIVE:To search for general patterns of the effects of autophagic mechanisms on skeletal muscle aging during exercise through a review of articles in this field. METHODS:(1)CNKI and Web of Science were searched,reviewed,and screened for relevant literature using the keywords of"Atg genes(proteins),autophagy,exercise,and skeletal muscle aging"to lay the theoretical foundation for the full-text analysis.(2)The comparative analysis method was used to compare the similarities and differences among the included documents to provide reasonable theoretical support for the arguments.By the further comparative analysis of the literature,the relationship between relevant indicators was clarified,to provide the ideas for the full-text analysis. RESULTS AND CONCLUSION:Atg family-mediated autophagy is indispensable for delaying skeletal muscle aging.Atg genes involved in regulating autophagy regulate the autophagic process in either a facilitative or inhibitory manner to improve the physiological morphology and function of skeletal muscle.Different exercise patterns,such as age,time,or intensity at initiation,may have heterogeneous effects on the expression of autophagy-related proteins,but long-term aerobic exercise regulates Atg-related proteins,induces skeletal muscle autophagy,and delays the loss of muscle mass.

The role and regulatory mechanism of RNA binding protein ZFP36in hypoxia/reoxygenation injury of cardiomyocytes / 西安交通大学学报(医学版)

【Objective】 To explore the role of ZFP36 in cardiomyocyte injury and autophagy induced by hypoxia/reoxygenation (H/R) so as to clarify its molecular regulatory mechanism. 【Methods】 H9C2 rat cardiomyocytes were infected with ZFP36 overexpressing lentivirus (OE-ZFP36) or its negative control lentivirus (OE-ZFP36 NC) to construct stable cell lines, respectively. Transfection of ATG4D overexpression plasmid (OE-ATG4D) improved the expression of ATG4D. Hypoxia/reoxygenation (H/R) induced myocardial cell injury. H9C2 cells were mainly divided into control group, H/R group, OE-ZFP36 NC+H/R group, OE-ZFP36+H/R group, OE-ATG4D NC+H/R group, OE-ATG4D+H/R group, OE-ZFP36+OE-ATG4D NC+H/R group, and OE-ZFP36+OE-ATG4D+H/R group. The protein expressions of ATG4D, Beclin1, LC3 and ZFP36 in H9C2 cells were detected by Western blotting. The mRNA levels of ZFP36 and ATG4D in H9C2 cells were detected by Real-time fluorescence quantitative PCR (qPCR). The viability of H9C2 cells was detected by CCK-8 assay. The levels of interleukin (IL-6) and tumor necrosis factor (TNF-α) in H9C2 cells were detected by enzyme-linked immunosorbent assay (ELISA). Reactive oxygen species (ROS) in H9C2 cells were detected by DCFH-DA method. SOD detection kit was used to detect the SOD level in H9C2 cells. The apoptosis of H9C2 cells was detected by flow cytometry. LC3 autophagosomes in H9C2 cells were detected by cellular immunofluorescence. Dual-luciferase reporter gene assay was used to detect the binding of ZFP36 and ATG4D mRNA in H9C2 cells. 【Results】 Compared with control group, H/R group showed decreased cell viability, increased IL-6 and TNF-α levels, increased ROS levels and decreased SOD levels, increased cell apoptosis. Up-regulated ATG4D and Beclin1 protein expression, increased LC3Ⅱ/LC3Ⅰ ratio, as well as upregulated ZFP36 expression were found in H/R group (all P<0.05). Compared with OE-ZFP36 NC+H/R group, elevated cell viability, decreased IL-6 and TNF-α levels, decreased ROS levels and increased SOD levels, reduced cell apoptosis (P<0.05), and downregulated ATG4D and Beclin1 protein expression, decreased LC3Ⅱ/LC3Ⅰ ratio were shown in OE-ZFP36+H/R group (all P<0.05). Compared with infection with OE-ZFP36 NC lentivirus, infection with OE-ZFP36 lentivirus decreased the luciferase activity of ATG4D 3′-UTR reporter gene, decreased the stability of ATG4D mRNA, and downregulated the H/R-induced ATG4D mRNA expression (all P<0.05). Compared with OE-ATG4D NC+H/R group, OE-ATG4D+H/R group had upregulated ATG4D mRNA and protein expression, decreased cell viability, increased IL-6 and TNF-α levels, increased ROS levels, decreased SOD levels and elevated cell apoptosis (all P<0.05). Compared with OE-ZFP36+OE-ATG4D NC+H/R group, OE-ZFP36+OE-ATG4D+H/R group had decreased cell viability, increased IL-6 and TNF-α levels, increased ROS levels, decreased SOD levels and elevated cell apoptosis (all P<0.05). 【Conclusion】 The expression of ZFP36 is upregulated in H/R-induced cardiomyocyte injury. The overexpression of ZFP36 inhibits H/R-induced cardiomyocyte injury and autophagy by regulating ATG4D, thus resisting cardiomyocyte H/R injury. It proves that ZFP36 is an important regulatory molecule against MI/RI.

In vitro activity comparison between anti-human T lymphocyte porcine immunoglobulin prepared by Cohn ethanol fractionation and ammonium sulphate precipitation and same type of rabbit-derived products / 中国生物制品学杂志

@#Objective To compare the in vitro activity of anti-human T lymphocyte porcine immunoglobulin(P-ATG)prepared by Cohn ethanol fractionation and ammonium sulphate precipitation,commercial rabbit anti-human thymocyte immunoglobulin(trade name:Thymoglobuline),and anti-human T lymphocyte rabbit immunoglobulin(trade name:Grafalon)so as to evaluate the properties of P-ATG prepared by two processes.Methods The four products were detected for the antibody-dependent cell-mediated cytotoxicity(ADCC)by lactate dehydrogenase(LDH)method,measured for the complement dependent cytotoxicity(CDC)by CCK-8 assay,and detected for the affinity for binding with different T cell antigens(CD3,CD4,CD8)by double immunofluorescence staining method.Results Among the four products,only Thymoglobuline at the high concentration(1 mg/mL)had a weak ADCC effect on human peripheral blood mononuclear cell(PBMC).All products could induce the CDC effect in human PBMC in a dose-dependent manner,among which the effect of Thymoglobuline was higher than that of P-ATG or Grafalon preared by two processes,about 3 to 4 times,and the effect of Grafalon was comparable to that of P-ATG.In the proportion of four products binding with T cell surface antigens CD3 and CD4 was similar,However the proportion of Thymoglobuline and Grafalon binding with CD8 antigen was slightly lower than that of P-ATG.Conclusion The in vitro activity of the P-ATG prepared by Cohn ethanol fractionation and ammonium sulphate precipitation was in good agreement,and was not lower than that of imported products at the clinical dose.

The Plant ATG8-binding Proteins / 生物化学与生物物理进展

ATG8-binding proteins play a key role in autophagy, selective autophagy or non-autophagy process by interacting between ATG8 and the ATG8-interacting motif (AIM) or the ubiquitin-interacting motif (UIM). There is great progress of ATG8-binding proteins in yeast and mammalian studies. However, the plant domain is still lagging behind. Therefore, the structure characteristics of plant ATG8 binding protein were firstly outlined. Unlike the single copy of ATG8 gene in yeast, many homologous genes have been identified in plant. The LIR/ AIM-docking site (LDS) of ATG8 protein contains W and L pockets and is responsible for binding to AIM. The ATG8 protein binds to UIM-containing proteins via UIM-docking site (UDS) instead of LDS. UDS is in the opposite position to LDS, so the ATG8 can bind both AIM and UIM proteins. Secondly, the structure and function of ATG8-binding proteins, especially the selective autophagy receptors, were systematically described. The protein NBR1 and Joka2, as proteaphagy receptors, guide ubiquitination protein aggregates to autophagosome for degradation by binding to AIM and ATG8 in Arabidopsis and tobacco, respectively. AtNBR1 also promotes plant immunity by binding the capsid protein of cauliflower mosaic virus and silencing suppressor HCpro of turnip mosaic virus, mediating pathogen autophagy. AtNBR1 still degrades chloroplast by microautophagy under photoinjure or chlorophagy during ibiotic stress. And the protein ORM mediates the degradation of plant immune receptor flagellin sensing 2 (FLS2) through AIM binding to ATG8. Interestingly, ATI1 and ATI2 participate in both chlorophagy and ERphagy. Otherwise, ER membrane protein AtSec62, soluble protein AtC53, and ubiquitin-fold modifier1-specific ligase 1 (UFL1) can be directly bound to ATG8 as ER autophagy receptors. As pexophagy receptor, AtPEX6 and AtPEX10 bind to ATG8 via AIM and participate in pexophagy. RPN10, as a 26S proteasome subunit, whose C-terminal UIM1 and UIM2 bind ubiquitin and ATG8, respectively, mediates the selective autophagy degradation of 26S proteasome inactivation when fully ubiquitinated. Plant-specific mitochondrial localization proteins FCS-like zinc finger (FLZ) and friendly (FMT) may also be mitophagy receptors. CLC2 binds to ATG8 via the AIM-LDS docking site and is recruited to autophagy degradation on the Golgi membrane. The tryptophan-rich sensory protein (TSPO) in Arabidopsis was involved in clearing free heme, porphyrin and plasma membrane intrinsic protein 2;7 (PIP2;7) through the combination of AIM and ATG8. The conformation of GSNOR1 changes during anoxia, exposing the interaction between AIM and ATG8, leading to selective degradation of GSNOR1. At last, the ATG8 binding proteins involved in autophagosome closure, transport and synthetic synthesis was summarized. For example, plant-specific FYVE domain protein required for endosomal sorting 1 (FREE1) is involved in the closure of autophagosomes during nutrient deficiency. Therefore, according to the recent research advances, the structure and function of plant ATG8-binding proteins were systematically summarized in this paper, in order to provide new ideas for the study of plant selective autophagy and autophagy.

Silencing GmATG10 results in activation of immune responses in soybean / 生物工程学报

Autophagy is a highly conserved mechanism for material degradation and recycling in eukaryote cells, and plays important roles in growth, development, stress tolerance and immune responses. ATG10 plays a key role in autophagosome formation. To understand the function of ATG10 in soybean, two homologous GmATG10 genes, namely GmATG10a and GmATG10b, were silenced simultaneously by bean pod mottle virus (BPMV) induced gene silencing. The carbon starvation induced by dark treatment and Western blotting analysis of GmATG8 accumulation level indicated that concurrent silencing GmATG10a/10b resulted in the impairment of autophagy in soybean; disease resistance and kinase assays demonstrated that GmATG10a/10b participated in the immune responses by negatively regulating the activation of GmMPK3/6, indicating that GmATG10a/10b plays a negative regulatory role in immune response in soybean.

Effect of autophagic core protein Atg101 on adipocyte senescence / 中华内分泌代谢杂志

Objective:To investigate the effect of autophagy related gene Atg101 on white adipocyte senescence.Methods:An Atg101 knockdown model of 3T3-L1 mature adipocytes was constructed to probe the effect of Atg101 on autophagy-related proteins LC3 and p62 protein. The RNA-seq database of human subcutaneous adipose tissue was constructed and analyzed, and the co-expressed gene set was predicted based on the pearson correlation coefficient( R2>0.4, P<0.05) between FPKM values of Atg101 and other gene, followed by KEGG and Reactome enrichment analysis. Young mouse(8 weeks old) and old mouse(18 months old) models were established, and the expression levels of Atg101 in inguinal white adipose tissue and epididymal white adipose tissue were detected by quantitative real-time PCR(RT-qPCR) and Western blot. Furthermore, the differences in white adipocyte senescence-associated secretory phenotype(SASP), cell cycle and mitochondrial homeostasis-related genes were detected by RNA-seq, Western blot, and RT-qPCR to analyze the effects of Atg101 silencing on adipocyte senescence. Results:The autophagy-related protein LC3-Ⅱ expression was significantly decreased and p62 protein was induced after Atg101 was knockdowned in 3T3-L1 adipocytes, suggesting impaired cell autophagy. KEGG enrichment analysis revealed that Atg101 co-expressed gene set was mainly enriched in autophagy and senescence-related pathways; Reactome enrichment analysis revealed that this gene set was associated with multiple cell cycle signaling pathways. RT-qPCR and Western blot confirmed that both mRNA and protein levels of Atg101 were down-regulated in inguinal white adipose tissue of aging mice, and protein levels in epididymal white adipose tissue were also significantly reduced. Finally, it was further confirmed that SASP-related genes were induced after Atg101 knockdown in white adipocytes, and cell cycle-specific gene expression was restricted and cytokine-dependent protein kinase inhibitors p16 and p21 expressions were significantly increased, while mitochondrial homeostasis regulatory genes were also suppressed.Conclusions:Knockdown of Atg101 may regulate white adipocyte senescence by inhibiting autophagic activity, presenting impaired mitochondrial homeostasis.

Effect of Zhiwei Fuwei Pills on autophagy in gastric antrum tissue of rats with precancerous lesions of gastric cancer based on mTOR/Beclin1/LC3 signaling axis / 中国临床药理学与治疗学

To investigate the effect of Zhiwei Fuwei Pills (ZWFW) on the expression of mammalian target of rapamycin (mTOR)/autophagy key molecule yeast Atg6 homologue (Beclin1)/microtubuleassociated protein 1 light chain 3 (LC3) signaling axis key molecules in gastric antrum tissue of rats with precancerous gastric lesions (PLGC). METHODS: SPF SD rats were randomly divided into normal group, model group, folic acid group, ZWFW low-dose, medium-dose, high-dose group. In addition to the normal group, the model group, folic acid group, ZWFW low-dose, medium-dose and high-dose groups, were used to establish the PLGC rat model by five factors compound modeling methods: N-methyl-N ' - nitro-n-nitroguanidine (MNNG) combined with hunger and satiation, ethanol intragastric administration, free drinking of ammonia and ranitidine feed. The rats were treated with normal saline, folic acid tablet aqueous solution (0.002 g/kg), ZWFW low-dose, medium-dose, high-dose aqueous solution (0.42, 0.84, 1.67 g/kg) for 4 weeks, and the stomach was removed by laparotomy. Hematoxylineosin (HE) staining was used to observe the histopathological changes in the antrum of rats, and real-time polymerase chain reaction (real-time PCR), Western blot (WB) and immunohistochemistry (IHC) were used to detect the expression of mammalian target of rapamycin mTOR, yeast Atg6 homologue 1 (Beclin1), microtubule-associated protein 1 light chain 3β (LC3B) mRNA and protein in the antrum of rats. RESULTS: Compared with the normal group, the Gastric antrum tissue of the model group was distended, thinner gastric wall, palegastric mucosa, atrophic and flat folds, disordered course and nodules and vegetations were visible. HE staining showed that compared with the normal group, the gastric mucosal glands in the model group were crowded and disordered, and the cell morphology was different, including a large number of goblet cells, basophilic cytoplasm, large, hyper-chromatic and irregular nuclei, and mucosal muscle infiltration and destruction. Compared with the model group, treated by ZWFW can significantly improve the pathological manifestations of gastric mucosal gland structure disorder and cell atypia. Compared with the normal group, mTOR mRNA and protein expression were significantly increased (P< 0.05) and Beclin1 and LC3B mRNA and protein expression were significantly decreased (P<0.05) in the antral tissue of rats in the model group; compared with the model group, mTOR mRNA and protein expression were decreased (P<0.05) in the medium and high dose groups of ZWFW, Beclin1 and LC3B protein expression in the antral tissue of rats in the low dose group of ZWFW and Beclin1 and LC3B mRNA and protein expression were increased (P<0.05) in the medium and high dose groups. CONCLUSION: Zhiwei Fuwei Pills can significantly improve the abnormal histopathological findings of gastric mucosa in PLGC model rats, and the mechanism may be related to the down-regulation of mTOR expression, up-regulation of Beclin1 and LC3B expression and then promoting autophagy.

Effect of mitophagy related genes on the antioxidant properties of Saccharomyces cerevisiae / 生物工程学报

Mitophagy is a process whereby cells selectively remove mitochondria through the mechanism of autophagy, which plays an important role in maintaining cellular homeostasis. In order to explore the effect of mitophagy genes on the antioxidant activities of Saccharomyces cerevisiae, mutants with deletion or overexpression of mitophagy genes ATG8, ATG11 and ATG32 were constructed respectively. The results indicated that overexpression of ATG8 and ATG11 genes significantly reduced the intracellular reactive oxygen species (ROS) content upon H2O2 stress for 6 h, which were 61.23% and 46.35% of the initial state, respectively. Notable, overexpression of ATG8 and ATG11 genes significantly increased the mitochondrial membrane potential (MMP) and ATP content, which were helpful to improve the antioxidant activities of the strains. On the other hand, deletion of ATG8, ATG11 and ATG32 caused mitochondrial damage and significantly decreased cell vitality, and caused the imbalance of intracellular ROS. The intracellular ROS content significantly increased to 174.27%, 128.68%, 200.92% of the initial state, respectively, upon H2O2 stress for 6 h. The results showed that ATG8, ATG11 and ATG32 might be potential targets for regulating the antioxidant properties of yeast, providing a new clue for further research.

Clinical Efficacy of Haplo-HSCT of ATG Combined with PTCy for Children with Myelodysplastic Syndrome / 中国实验血液学杂志

OBJECTIVE@#To investigate the efficacy and safety of haploidentical hematopoietic stem cell transplantation (haplo-HSCT) in combination of ATG and post-transplant cyclophosphamide (PTCy) -induced immune tolerance after transplantation in treatment of childhood myelodysplastic syndromes(MDS）.@*METHODS@#From July 2016 to November 2020, a total of 8 children with MDS receiving the haploidentical allo-HSCT combined with ATG and PTCy-induced immune tolerance after transplantation in our hospital were enrolled, whose clinical data were retrospected and analyzed.@*RESULTS@#Median age at diagnosis of the 8 children (1 male and 7 females) was 6.4 (range, 10 months to 15 years) years old. The median medical history of MDS was 2.7 years (range, 3 months to 8 years). Among the 8 patients, 7 cases were diagnosed with refractory cytopenia of childhood and one with refractory anemia with excess of blasts. The HSC donors were father, mother or brother of patients and HLA matching in 6-9/12 loci were identical. All the donors were healthy and didn't carry the same pathogenic genes as the recipients. The median age of donors was 36.4 (range, 25 to 49) years old. The median mononuclear cell (MNC) number of the graft was 19.8, ranging in (13.2-47.3)×108/kg, and the median CD34+ cell number was 11.8×106/kg, ranging in (5.0-18.3)×106/kg. Graft-versus-host disease prophylactic regimen was started on day 3 and 4 after transplantation, in which cyclophosphamide (50 mg/kg·d) was administered by intravenous infusion. From day 5 after transplantation, low-dose tacrolimus was administered by intravenous infusion and mycophenolate mofetil was administered orally. The median time of neutrophil and platelet engraftment was 12.6 (rang, 11 to 15) days and 13.3 (rang, 11 to 18) days, respectively. All the patients achieved full donor chimerism on neutrophil engraftment after transplantation. The median follow-up time was 1 032 (rang, 747 to 1 536) days. Both overall survival rate and disease-free survival rate were 100%.@*CONCLUSION@#Haplo-HSCT combined with ATG and PTCy-induced immune tolerance after transplantation is a safe and effective treatment for children with MDS.

MicroRNA-34c-5p provokes isoprenaline-induced cardiac hypertrophy by modulating autophagy via targeting ATG4B

Pathological cardiac hypertrophy serves as a significant foundation for cardiac dysfunction and heart failure. Recently, growing evidence has revealed that microRNAs (miRNAs) play multiple roles in biological processes and participate in cardiovascular diseases. In the present research, we investigate the impact of miRNA-34c-5p on cardiac hypertrophy and the mechanism involved. The expression of miR-34c-5p was proved to be elevated in heart tissues from isoprenaline (ISO)-infused mice. ISO also promoted miR-34c-5p level in primary cultures of neonatal rat cardiomyocytes (NRCMs). Transfection with miR-34c-5p mimic enhanced cell surface area and expression levels of foetal-type genes atrial natriuretic factor (Anf) and β-myosin heavy chain (β-Mhc) in NRCMs. In contrast, treatment with miR-34c-5p inhibitor attenuated ISO-induced hypertrophic responses. Enforced expression of miR-34c-5p by tail intravenous injection of its agomir led to cardiac dysfunction and hypertrophy in mice, whereas inhibiting miR-34c-5p by specific antagomir could protect the animals against ISO-triggered hypertrophic abnormalities. Mechanistically, miR-34c-5p suppressed autophagic flux in cardiomyocytes, which contributed to the development of hypertrophy. Furthermore, the autophagy-related gene 4B (ATG4B) was identified as a direct target of miR-34c-5p, and miR-34c-5p was certified to interact with 3' untranslated region of Atg4b mRNA by dual-luciferase reporter assay. miR-34c-5p reduced the expression of ATG4B, thereby resulting in decreased autophagy activity and induction of hypertrophy. Inhibition of miR-34c-5p abolished the detrimental effects of ISO by restoring ATG4B and increasing autophagy. In conclusion, our findings illuminate that miR-34c-5p participates in ISO-induced cardiac hypertrophy, at least partly through suppressing ATG4B and autophagy. It suggests that regulation of miR-34c-5p may offer a new way for handling hypertrophy-related cardiac dysfunction.

Autophagy enhanced by curcumin ameliorates inflammation in atherogenesis via the TFEB-P300-BRD4 axis

Disturbance of macrophage-associated lipid metabolism plays a key role in atherosclerosis. Crosstalk between autophagy deficiency and inflammation response in foam cells (FCs) through epigenetic regulation is still poorly understood. Here, we demonstrate that in macrophages, oxidized low-density lipoprotein (ox-LDL) leads to abnormal crosstalk between autophagy and inflammation, thereby causing aberrant lipid metabolism mediated through a dysfunctional transcription factor EB (TFEB)-P300-bromodomain-containing protein 4 (BRD4) axis. ox-LDL led to macrophage autophagy deficiency along with TFEB cytoplasmic accumulation and increased reactive oxygen species generation. This activated P300 promoted BRD4 binding on the promoter regions of inflammatory genes, consequently contributing to inflammation with atherogenesis. Particularly, ox-LDL activated BRD4-dependent super-enhancer associated with liquid-liquid phase separation (LLPS) on the regulatory regions of inflammatory genes. Curcumin (Cur) prominently restored FCs autophagy by promoting TFEB nuclear translocation, optimizing lipid catabolism, and reducing inflammation. The consequences of P300 and BRD4 on super-enhancer formation and inflammatory response in FCs could be prevented by Cur. Furthermore, the anti-atherogenesis effect of Cur was inhibited by macrophage-specific Brd4 overexpression or Tfeb knock-out in Apoe knock-out mice via bone marrow transplantation. The findings identify a novel TFEB-P300-BRD4 axis and establish a new epigenetic paradigm by which Cur regulates autophagy, inhibits inflammation, and decreases lipid content.

Effect of Danggui Niantongtang on Expression of Autophagy-related Proteins LC3， Beclin1 and p62 in Rats with Adjuvant Arthritis Differentiated into Wind-damp-heat Impediment / 中国实验方剂学杂志

ObjectiveTo observe the preventive and control effects of Danggui Niantongtang against adjuvant arthritis differentiated into wind-damp-heat impediment in rats and its influences on the expression of autophagy-related proteins microtubule-associated protein 1 light chain 3 （LC3）， homolog of yeast Atg6 （Beclin1） and p62. MethodThe six-week-old male SD rats were randomly divided into the normal group， wind-damp-heat impediment model group， low-， medium-， and high-dose Danggui Niantongtang （5.67， 11.34， 22.68 g·kg-1） groups， and methotrexate （MTX， 1.35 mg·kg-1） group， with 10 rats in each group. A rat model of adjuvant arthritis was established by subcutaneous injection of inactivated Mycobacterium tuberculosis into the tail root， followed by exposure to the manual climatic box for 16 d for inducing the wind-damp-heat impediment. The drugs were administered intragastrically on the day of immunization for 28 d. The general conditions of rats were observed and the swelling degree of toes and arthritis index （AI） were detected. The pathological changes in the synovial tissues of the knee joints were observed by hematoxylin-eosin （HE） staining. The mRNA expression levels of LC3， Beclin1， and p62 in the synovial tissues were measured by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）， followed by the assay of their protein expression by Western blot and immunohistochemistry. ResultCompared with the normal group， the wind-damp-heat impediment model group exhibited significantly increased swelling degree of toes （P<0.01）， increased AI （P<0.01）， proliferated synovial cells （P<0.01）， up-regulated LC3 and Beclin1 protein and mRNA expression （P<0.01）， and down-regulated p62 protein and mRNA expression （P<0.01） after 16， 20， 24， 28-d medication. Compared with the wind-damp-heat impediment model group， each medication group displayed alleviated toe swelling and synovial hyperplasia to different degrees， decreased mRNA and protein expression levels of LC3 and Beclin1 （P<0.01）， and increased p62 mRNA and protein expression （P<0.05，P<0.01）， with the best outcomes observed in the medium-dose Danggui Niantongtang group. ConclusionDanggui Niantongtang effectively relieves adjuvant arthritis due to wind-damp-heat impediment in rats， which may be related to its regulation of the expression of autophagy-related proteins LC3， Beclin1， and p62 and the inhibition of autophagy.

Effect of Qiyu Sanlong Decoction in Inducing Autophagy of A549 Cells by Regulating Molecular Expression Related to Signal Axis of mTOR-Beclin1-LC3 / 中国实验方剂学杂志

Objective:To observe the effect of Qiyu Sanlong decoction （QYSL） on the expressions of key molecules in signal axis of mammalian rapamycin target protein （mTOR）/yeast Atg6 homologous （Beclin1）/ microtubule-associated protein1 light chain3 （LC3） in A549 cells. Method:With A549 cells as the research object， the effect of QYSL medicated serum on cell viability of A549 cells were detected by cell counting kit-8 （CCK-8） method. The effect of QYSL decoction on A549 cell apoptosis， autophagosome formation and the expression of autophagy markers were detected by Terminal-deoxynucleoitidyl transferase mediated nick end labeling （TUNEL） method， transmission electron microscope （TEM）， Real-time polymerase chain reaction （Real-time PCR） and Western blot. Result:QYSL medicated serum could inhibit the viability of A549 cells in a concentration-dependent manner. Compared with the blank serum group， the number of apoptotic A549 cells in the QYSL medicated serum group was significantly increased （P<0.01）， and the formation of autophagosome was significantly increased. Compared with the blank serum group， the mRNA and protein expressions of mTOR in A549 cells in the QYSL serum group were significantly decreased （P<0.01）， while mRNA and protein expressions of Beclin-1， autophagy related genes 5 （ATG5）， autophagy related genes 13 （ATG13） were significantly increased （P<0.01）. Conclusion:QYSL decoction can induce autophagy in A549 cells， and its specific mechanism may be related to the down-regulation of mTOR expression， the up-regulation of Beclin1， ATG5， ATG13 and LC3 expression， and the promotion of LC3Ⅰ conversion to LC3Ⅱ.

Multi-omics approaches identify

Autophagy is a critical cellular homeostatic mechanism, and its dysfunction is linked to invasive breast carcinoma (BRCA). Recently, several omics methods have been applied to explore autophagic regulators in BRCA; however, more reliable and robust approaches for identifying crucial regulators and druggable targets remain to be discovered. Thus, we report here the results of multi-omics approaches to identify potential autophagic regulators in BRCA, including gene expression (EXP), DNA methylation (MET) and copy number alterations (CNAs) from The Cancer Genome Atlas (TCGA). Newly identified candidate genes, such as

Lipids and membrane-associated proteins in autophagy

Autophagy is essential for the maintenance of cellular homeostasis and its dysfunction has been linked to various diseases. Autophagy is a membrane driven process and tightly regulated by membrane-associated proteins. Here, we summarized membrane lipid composition, and membrane-associated proteins relevant to autophagy from a spatiotemporal perspective. In particular, we focused on three important membrane remodeling processes in autophagy, lipid transfer for phagophore elongation, membrane scission for phagophore closure, and autophagosome-lysosome membrane fusion. We discussed the significance of the discoveries in this field and possible avenues to follow for future studies. Finally, we summarized the membrane-associated biochemical techniques and assays used to study membrane properties, with a discussion of their applications in autophagy.

Natural compounds modulate the autophagy with potential implication of stroke

Stroke is considered a leading cause of mortality and neurological disability, which puts a huge burden on individuals and the community. To date, effective therapy for stroke has been limited by its complex pathological mechanisms. Autophagy refers to an intracellular degrading process with the involvement of lysosomes. Autophagy plays a critical role in maintaining the homeostasis and survival of cells by eliminating damaged or non-essential cellular constituents. Increasing evidence support that autophagy protects neuronal cells from ischemic injury. However, under certain circumstances, autophagy activation induces cell death and aggravates ischemic brain injury. Diverse naturally derived compounds have been found to modulate autophagy and exert neuroprotection against stroke. In the present work, we have reviewed recent advances in naturally derived compounds that regulate autophagy and discussed their potential application in stroke treatment.

Lonicerin targets EZH2 to alleviate ulcerative colitis by autophagy-mediated NLRP3 inflammasome inactivation

Aberrant activation of NLRP3 inflammasome in colonic macrophages strongly associates with the occurrence and progression of ulcerative colitis. Although targeting NLRP3 inflammasome has been considered to be a potential therapy, the underlying mechanism through which pathway the intestinal inflammation is modulated remains controversial. By focusing on the flavonoid lonicerin, one of the most abundant constituents existed in a long historical anti-inflammatory and anti-infectious herb

Chikusetsu saponin Ⅳa ameliorates myocardial hypertrophy of rats through regulating expression of miR199a-5p/Atg5 / 中国中药杂志

The present study investigated the effects of chikusetsu saponin Ⅳa(CHS Ⅳa) on isoproterenol(ISO)-induced myocardial hypertrophy in rats and explored the underlying molecular mechanism. ISO was applied to establish a rat model of myocardial hypertrophy, and CHS Ⅳa(5 and 15 mg·kg~(-1)·d~(-1)) was used for intervention. The tail artery blood pressure was measured. Cardiac ultrasound examination was performed. The ratio of heart weight to body weight(HW/BW) was calculated. Morphological changes in the myocardial tissue were observed by HE staining. Collagen deposition in the myocardial tissue was observed by Masson staining. The mRNA expression of myocardial hypertrophy indicators(ANP and BNP), autophagy-related genes(Atg5, P62 and beclin1), and miR199 a-5 p was detected by qRT-PCR. Atg5 protein expression was detected by Western blot. The results showed that the model group exhibited increased tail artery blood pressure and HW/BW ratio, thickened left ventricular myocardium, enlarged myocardial cells, disordered myocardial fibers with widened interstitium, and a large amount of collagen aggregating around the extracellular matrix and blood vessels. ANP and BNP were largely expressed. Moreover, P62 expression was up-regulated, while beclin1 expression was down-regulated. After intervention by CHS Ⅳa at different doses, myocardial hypertrophy was ameliorated and autophagy activity in the myocardial tissue was enhanced. Meanwhile, miR199 a-5 p expression declined and Atg5 expression increased. As predicted by bioinformatics, Atg5 was a target gene of miR199 a-5 p. CHS Ⅳa was capable of preventing myocardial hypertrophy by regulating autophagy of myocardial cells through the miR-199 a-5 p/Atg5 signaling pathway.

Effect of total saponins from Panax japonicus on non-alcoholic steatohepatitis by regulating autophagy / 中国中药杂志

Non-alcoholic steatohepatitis(NASH) was induced by high-sugar and high-fat diet in mice to investigate the intervention effect of total saponins from Panax japonicus(TSPJ) and explore its possible mechanism. Mice were fed with high-sugar and high-fat diet to establish NASH model, and intervened with different doses of TSPJ(15, 45 mg·kg~(-1)). The animals were fed for 26 weeks. The histomorphology and pathological changes of liver tissues were observed by HE staining. The transcriptional expression levels of miR-199 a-5 p, autophagy related gene 5(ATG5) and inflammatory cytokines interleukin-6(IL-6), interleukin-1β(IL-1β) and tumor necrosis factor α(TNF-α) in mouse liver were measured by quantitative Real-time polymerase chain reaction(qRT-PCR). Western blot was used to detect the expression of autophagy-related proteins ATG5, P62/SQSTM1(P62), and microtubule-associated protein light chain 3(LC3)-I/Ⅱ proteins in mouse liver. The expression of P62 protein was detected by immunofluorescence staining. In order to verify the targeting regulation relationship between miR-199 a-5 p and ATG5, miR mimic/inhibitor NC and miR-199 a-5 p mimic/inhibitor were transfected into Hepa 1-6 cells, and the expression of ATG5 mRNA and protein was detected. pMIR-reportor ATG5-3'UTR luciferase reporter gene plasmid was constructed and co-transfected with miR mimic/inhibitor NC and miR-199 a-5 p mimic/inhibitor into Hepa 1-6 cells to detect luciferase activity. In vivo, HE staining in the model group showed typical fatty degeneration and inflammatory infiltration, with increased expression of miR-199 a-5 p and decreased expression of ATG5 mRNA and protein. The expression of autophagy-associated protein P62 increased significantly, the ratio of LC3Ⅱ/Ⅰ decreased, and the transcriptional expression of inflammatory factors increased significantly. After the intervention by TSPJ, the pathological performance of liver tissue was significantly improved, the expression of miR-199 a-5 p decreased and the expression of ATG5 mRNA and protein increased, the expression of autophagy-associated protein P62 decreased significantly, the ratio of LC3Ⅱ/Ⅰ increased, and the transcriptional expression of inflammatory cytokines IL-6, IL-1β and TNF-α decreased significantly. In vitro, it was found that the expression of ATG5 mRNA and protein and luciferase activity decreased significantly in miR-199 a-5 p overexpression cells, while after inhibition of miR-199 a-5 p expression, the expression level of ATG5 mRNA and protein and luciferase activity increased. The results showed that TSPJ can improve NASH in mice fed with high-sugar and high-fat diet, and its mechanism may be related to the regulation of miR-199 a-5 p/ATG5 signal pathway, the regulation of autophagy activity and the improvement of inflammatory response of NASH.