Level Of Autophagy In Cells Research Articles

Increased DNMT1 Involvement in the Activation of LO2 Cell Death Induced by Silver Nanoparticles via Promoting TFEB-Dependent Autophagy.

The accumulation of exogenous silver nanoparticles (AgNPs) will terminally bring about liver injury, including cell death, where DNA methylation tends to be a crucial epigenetic modulator. The change in the cell autophagy level verified to be closely associated with hepatocyte death has been followed with wide interest. But the molecular toxicological mechanisms of AgNPs in relation to DNA methylation, autophagy, and cell death remain inconclusive. To address the issue above, in LO2 cells treated with increasing concentrations of AgNPs (0, 5, 10, and 20 μg/mL), a cell cytotoxicity assay was performed to analyze the level of cell death, which also helped to choose an optimal concentration for next experiments. An immunofluorescence assay was used to determine the autophagic flux as well as TFEB translocation, with qRT-PCR and western blot being used to analyze the expression level of autophagy-related genes and proteins. According to our findings, in the determination of cell viability, 20 μg/mL (AgNPs) was adopted as the best working concentration. LO2 cell death, autophagy, and TFEB nuclear translocation were induced by AgNPs, which could be inhibited by lysosome inhibitor chloroquine (CQ) or siRNA specific for TFEB. Moreover, AgNP exposure led to DNA hypermethylation, with DNMT1 taking part mainly, which could be obviously prevented by 5-Aza-2'-deoxycytidine (5-AzaC) or trichostatin A (TSA) treatment or DNMT1 knockout in LO2 cells. Our studies suggest that through TFEB-dependent cell autophagy, increased DNMT1 may facilitate cell death induced by AgNPs.

Matrix stiffness regulates neovascular homeostasis through autophagy in nude mice.

One of the major obstacles to the effective application of vascularized fruit is an insufficient understanding of the relationship between the microenvironment and neovascular homeostasis. The role of extracellular matrixstiffness in regulating the structural and functional stability of neovascularization has not yet been elucidated. This study explored the effects of matrix stiffness on neovascular homeostasis in nude mice. Dextran hydrogels with three different stiffnesses were separately combined with mouse bone marrow-derived endothelial progenitor cells (EPCs) and subcutaneously implanted into the backs of nude mice. After 14 days, neovascular homeostasis indicators in the different groups were measured. Cell autophagy levels were evaluated, and inhibitor assays were performed to explore the underlying mechanism. New blood vessels were generated in the three stiffnesses of the EPC-loaded dextran hydrogels 14 days after implantation. The newly formed vessels tended to have better structural stability in softer hydrogels. Endothelial function markers, such as endothelial nitric oxide synthaseand E-selectin, were downregulated as the matrix stiffness increased. Furthermore, we found that cell autophagy levels decreased in stiffer matrices, and autophagy inhibition attenuated neovascular homeostasis. A soft matrix is conducive to maintaining neovascular homeostasis through autophagy in nude mice.

Morin induces autophagy and apoptosis in hepatocellular carcinoma cells through Akt/mTOR/STAT3 pathway

This study investigated the effect and mechanism of morin in inducing autophagy and apoptosis in hepatocellular carcinoma cells through the protein kinase B(Akt)/mammalian target of rapamycin(mTOR)/signal transducer and activator of transcription protein 3(STAT3) pathway. Human hepatocellular carcinoma SK-HEP-1 cells were stimulated with different concentrations of morin(0, 50, 100, 125, 200, and 250 μmol·L~(-1)). The effect of morin on the viability of SK-HEP-1 cells was detected by Cell Counting Kit-8(CCK-8). The effect of morin on the proliferation and apoptosis of SK-HEP-1 cells was investigated using colony formation assay, flow cytometry, and BeyoClick~(TM) EdU-488 with different concentrations of morin(0, 125, and 250 μmol·L~(-1)). The changes in the autophagy level of cells treated with morin were examined by transmission electron microscopy and autophagy inhibitors. The impact of morin on the expression levels of proteins related to the Akt/mTOR/STAT3 pathway was verified by Western blot. Compared with the control group, the morin groups showed decreased viability of SK-HEP-1 cells in a time-and concentration-dependent manner, increased number of apoptotic cells, up-regulated expression level of apoptosis marker PARP, up-regulated phosphorylation level of apoptosis-regulating protein H2AX, decreased number of positive cells and the colony formation rate, an upward trend of expression levels of autophagy-related proteins LC3-Ⅱ, Atg5, and Atg7, and decreased phosphorylation levels of Akt, mTOR, and STAT3. These results suggest that morin can promote apoptosis, inhibit proliferation, and induce autophagy in hepatocellular carcinoma cells, and its mechanism of action may be related to the Akt/mTOR/STAT3 pathway.

Corn Silk Polysaccharides with Different Carboxyl Contents Reduce the Oxidative Damage of Renal Epithelial Cells by Inhibiting Endocytosis of Nano-calcium Oxalate Crystals.

Renal epithelial cell injury and cell-crystal interaction are closely related to kidney stone formation. This study aims to explore the inhibition of endocytosis of nano-sized calcium oxalate monohydrate (nano-COM) crystals and the cell protection of corn silk polysaccharides (CCSPs) with different carboxyl contents (3.92, 7.75, 12.90, and 16.38%). The nano-COM crystals protected or unprotected by CCSPs were co-cultured with human renal proximal tubular epithelial cells (HK-2), and then the changes in the endocytosis of nano-COM and cell biochemical indicators were detected. CCSPs could inhibit the endocytosis of nano-COM by HK-2 cells and reduce the accumulation of nano-COM in the cells. Under the protection of CCSPs, cell morphology is restored, intracellular superoxide dismutase levels are increased, lipid peroxidation product malondialdehyde release is decreased, and mitochondrial membrane potential and lysosomal integrity are increased. The release of Ca2+ ions in the cell, the level of cell autophagy, and the rate of cell apoptosis and necrosis are also reduced. CCSPs with higher carboxyl content have better cell protection abilities. CCSPs could inhibit the endocytosis of nano-COM crystals and reduce cell oxidative damage. CCSP3, with the highest carboxyl content, shows the best biological activity.

2,3,5,4'-Tetrahydroxystilbene (TG1), a Novel Compound Derived from 2,3,5,4'-Tetrahydroxystilbene-2-O-β-D-glucoside (THSG), Inhibits Colorectal Cancer Progression by Inducing Ferroptosis, Apoptosis, and Autophagy.

Colorectal cancer (CRC) is one of the deadliest cancers worldwide and long-term survival is not guaranteed in metastatic disease despite current multidisciplinary therapies. A new compound 2,3,5,4'-Tetrahydroxystilbene (TG1), derived from THSG (2,3,5,4'-Tetrahydroxystilbene-2-O-β-D-Glucoside), has been developed, and its anticancer ability against CRC is verified in this study. HCT116, HT-29, and DLD-1 were treated with TG1 and the IC50 was measured using a sulforhodamine B assay. A Xenograft mouse model was used to monitor tumor growth. Apoptosis and autophagy, induced by TG1 in CRC cells, were examined. RNA-sequencing analysis of CRC cells treated with TG1 was performed to discover underlying pathways and mechanisms. The results demonstrated that treatment with TG1 inhibited CRC proliferation in vitro and in vivo and induced apoptotic cell death, which was confirmed by Annexin V-FITC/PI staining and Western blotting. Additionally, TG1 treatment increased the level of autophagy in cells. RNA-sequencing and GSEA analyses revealed that TG1 was associated with MYC and the induction of ferroptosis. Furthermore, the ferroptosis inhibitor Bardoxolone abrogated the cytotoxic effect of TG1 in CRC cells, indicating that ferroptosis played a crucial role in TG1-induced cytotoxicity. These findings suggest that TG1 might be a potential and potent compound for clinical use in the treatment of CRC by inhibiting proliferation and inducing ferroptosis through the MYC pathway.

Piceatannol-3′-O-β-D-glucopyranoside attenuates colistin-induced neurotoxicity by suppressing oxidative stress via the NRF2/HO-1 pathway

BackgroundMultidrug-resistant Gram-negative bacteria are the most pressing problem in treating infectious diseases. As one of the primary drugs for multidrug-resistant Gram-negative bacteria, the neurotoxicity of colistin has become a significant challenge in clinical practice. PurposeThis study aimed to investigate the potential effect of piceatannol-3′-O-β-D glucopyranoside (PG) on colistin-induced neurotoxicity and the underlying mechanism. MethodsIn vitro, nerve cell damage models were established by exposing N2a cells to 400 μM colistin for 24 h. The effects of PG on cell viability, apoptosis level, and oxidative stress level were analyzed. A western blot experiment was performed to determine the NRF2 pathway, apoptosis, and autophagy-related proteins. Mitochondrial morphology and mitochondrial membrane potential were detected after staining using laser confocal microscopy. In vivo, nerve injury mouse model was established by intracerebroventricular colistin administration. Morphological changes in brain tissues were observed using HE and Nissl staining. ResultsPG significantly reduced colistin-induced neuronal apoptosis levels. The apoptosis-related protein expressions were suppressed after PG intervention. Mechanistically, PG increased the levels of antioxidant factors and decreased the levels of oxidative factors, which might be related to the activation of the NRF2 pathway. In addition, PG treatment reversed the deviations in mitochondrial morphology and membrane potential. PG suppressed autophagy levels in N2a cells, possibly because PG inhibited colistin-induced apoptosis, thus reducing the level of spontaneous protective autophagy in cells. Nrf2 knockdown N2a cell models were applied to confirm that the activation of the NRF2 pathway played a vital role in PG alleviating the nerve damage caused by colistin. ConclusionPG is a potential treatment option for colistin-induced neurotoxicity. It mitigated colistin-induced oxidative stress-associated injury and mitochondrial damage by activating the NRF2/HO-1 pathway, thus reducing nerve cell apoptosis.

Bushen Wenyang Huayu Decoction Targets TLR4/NF-κB Mediated Autophagy to Treat Endometriosis Effectively.

Endometriosis has been found to be closely related to autophagy. This study aimed to elucidate the possible mechanism of Bushen Wenyang Huayu Decoction (BWHD) in treating endometriosis (EMs) by targeting TLR4/NF-κB-mediated autophagy. Autologous grafting was used to generate the EMs model in rats. Once the model was developed, BWHD high-dose and low-dose groups received intragastric administration of BWHD, and the gestrinone group served as a positive control. Immunofluorescence labeling and Western blotting were used for the protein expression of toll-like receptor 4 (TLR4), nuclear transcription factor-κB (NF-κB), Beclin-1, and selective autophagy connector protein P62 (P62). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze mRNA levels of TLR4, NF-κB, Beclin-1, and P62. We found that BWHD significantly reduced the size of ectopic lesions in rats with EMs, regulated reproductive hormone levels, and alleviated the cell autophagy level. It suggested that BWHD could be an effective treatment of EMs by targeting TLR4/NF-κB signaling pathway.

DR1-CSE/H2S pathway upregulates autophagy and inhibits H9C2 cells damage induced by high glucose

In the cardiovascular system, long-term high glucose (HG) can lead to cardiomyocyte damage. Hydrogen sulfide (H2S) reduces cell autophagy in cardiomyocytes. Dopamine 1 receptors (DR1), a specific binding receptor for dopamine, which has a significant regulatory effect on cardiomyocytes. However, it is unclear whether DR1 inhibits HG-induced cardiomyocyte damage by regulating endogenous H2S production and the level of cell autophagy. The present data indicated that the expression of DR1 and cystathionine-γ-lyase (CSE, a key enzyme for endogenous H2S production) and H2S content were significantly reduced in HG-induced cardiomyocytes, which was reversed by SKF38393 (an agonist of DR1). NaHS (an exogenous H2S donor) only increased H2S content and the expression of CSE with no effect on DR1 expression. HG reduced cell viability, the expression of Bcl-2 and Beclin1, the production of autophagosomes and LC3 II/I ratio and increased the cell apoptotic ratio, the expression of cleaved caspase-3, cleaved caspase-9, cytochrome c, P62, and p-mTOR/t-mTOR ratio. SKF38393 and NaHS reversed the effects of HG. PPG (an inhibitor of CSE) and 3MA (an inhibitor of autophagy) abolished the beneficial effect of SKF38393. In addition, AICAR (an agonist of AMPK) and Rapamycin (an inhibitor of mTOR) increased the production of autophagosomes but decreased the p-mTOR/t-mTOR ratio, which was similar to the effects of SKF38393 and 3MA. Our findings suggest that DR1 reduces the HG-induced cardiomyocyte damage via up-regulating the CSE/H2S pathway, which increases cell autophagy by inhibiting the activation of mTOR.

Abstract LB133: EGFR upregulation in breast cancer cells: A key mechanism of escape from antiestrogen induced death, autophagy, and senescence

Abstract EGFR is upregulated commonly in multiple cancer types, including breast cancer. However, the multiple role(s) of EGFR in breast cancer are not fully elucidated. In this study, we characterized a unique antiestrogen resistant breast cancer subline and provide compelling data that upregulation of the EGFR/MEK1/MAPK1/2 signaling axis during antiestrogen treatment facilitated escape from senescence, autophagy, and BimEL-dependent apoptosis. Our studies were conducted with the antiestrogen resistant TR5 cell line established by our laboratory, using a step-wise drug selection with the antiestrogen 4-hydroxytamoxifen (4-OHT) of the estrogen receptor α (ER+) MCF-7 breast cancer cells. A preliminary microarray comparing mRNA expression between 4-OHT-selected TR5 cells and MCF-7 parent cells identified upregulation of the epidermal growth factor receptor (EGFR). Western blot showed a greater than 5-fold upregulation of EGFR expression and downregulation of estrogen receptor alpha (ERα) compared to the levels in MCF-7 and T47-D (antiestrogen sensitive). Selective inhibition of EGFR, phosphorylated at Tyr 1068, was achieved with erlotinib. Inhibition blocked MEK1/MAPK1/2 signaling, which was also upregulated in 4-OHT selected TR5 cells, with detectable increases in the pro-apoptotic BH3 protein BimEL, and a fifty percent reduction in cell viability. However, a high number of cells survived erlotinib therapy due to autophagy induction determined to be cytoprotective. Autophagy induction was identified by analyzing the expression/turnover of autophagy proteins LC3-II and p62, a standard approach to ascertain autophagy levels in cells. Further, erlotinib treatment induced a 2-fold increase in the number of senescent cells in the surviving population. Senescent cells were identified by β-galactosidase staining of erlotinib-treated cells utilizing confocal microscopy. Interestingly, blockade of autophagy with the lysosomotrophic compound chloroquine enhanced the senescence cell population, suggesting that either autophagy blocked senescence induction or lysosome impairment increased senescence. Mechanistically, we have identified phosphorylated AMP-activated protein kinase (pAMPK) as being elevated under conditions of erlotinib treatment, and reduced in 4-OHT-selected TR5 cells relative to MCF-7 parent cells. This correlation supports the hypothesis that pAMPK was downregulated during 4-OHT selection of TR5 cells by elevated EGFR/MEK/MAPK1/2 signaling to allow surviving cells escape senescence induction and/or anti-proliferative autophagy. Overall we propose that EGFR mediated downregulation of pAMPK is a major regulatory pathway in breast cancer cells surviving endocrine treatment. Our current studies are dissecting the role of EGFR-mediated AMPK regulation with somatic cell-based studies and pharmacological intervention. Citation Format: Mackenzie L. Hagan, Michael McGrath, Carol Joseph, Allison Lewis, John R. Barrett, Muthusamy Thangaraju, David Gewirtz, Patricia V. Schoenlein. EGFR upregulation in breast cancer cells: A key mechanism of escape from antiestrogen induced death, autophagy, and senescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB133.

SIRT2 Affects Cell Proliferation and Apoptosis by Suppressing the Level of Autophagy in Renal Podocytes

Purpose Despite the discovery of many important molecules in diabetic nephropathy, there has been very limited progress in the management of diabetic kidney diseases and the design of new drugs. To fill this gap, the present study explored the expression of SIRT2 in high-glucose murine kidney foot cells and its impact on cell biological functions. Methods Expression levels of SIRT2 in the MPC-5 of murine kidney foot cells after high and normal glucose treatment or in cells targeted with siRNA were detected using qRT-PCR. Cellular proliferation and programmed cell death were analyzed via the CCK8 assay and flow cell technique, separately. Levels of autophagy markers were measured by western blotting, and chloroquine treatment was applied to the cells to observe the effect of SIRT2 on cell proliferation and apoptosis after treatment. Results The expression level of SIRT2 was remarkably upregulated in the high-GLU group in contrast to the low-GLU group. The cell proliferation and autophagy levels were significantly reduced, and apoptosis was remarkably reinforced in the high-GLU group in contrast to the normal GLU group. However, knocking down the expression level of SIRT2 caused an increase in cell proliferation and cell autophagy levels and significantly weakened apoptosis. Chloroquine influenced cell proliferation and apoptosis in cells targeted with SIRT2 siRNA. Conclusion SIRT2 expression was upregulated in hyperglycaemic murine kidney foot cells, and knocking down the expression level of SIRT2 affected the biological function of the cells. We found that SIRT2 may modulate cell proliferation and apoptosis by regulating cell autophagy.

Xuanfei Pingchuan Capsules Ameliorate Autophagy in Human Bronchial Epithelial Cells by Inhibiting p38 Phosphorylation.

Background: This study aimed to investigate the protective effect of Xuanfei Pingchuan Capsules (XFPC) on autophagy and p38 phosphorylation in human bronchial epithelial (HBE) cells induced by cigarette smoke extract (CSE). Methods: HBE cells were divided into five groups: blank, CSE, low XFPC dose (XFPC-L), medium XFPC dose (XFPC-M), and high XFPC dose (XFPC-H). HBE cells were induced by CSE to establish a cell model for chronic obstructive pulmonary disease, and different doses of XFPC medicated serum were used to treat the cells. The Cell Counting Kit-8 was used to detect cell viability. Flow cytometry was used to detect cell apoptosis. Fluorescence microscopy and the expression level of microtubule-associated protein light chain 3 (LC3)-II in immunohistochemical method were used to observe autophagy in cells. Western blot was used to detect the protein expression level of p38, phospho-p38 (p-p38), LC3-I, LC3-II and Beclin 1. Real-time polymerase chain reaction was used to detect the expression of LC3-I, LC3-II and Beclin 1 on mRNA level. Results: Compared with the blank group, the cell viability of the CSE group was significantly decreased, and apoptosis and the level of autophagy in cells were significantly increased. The mRNA and protein expression of LC3-I, LC3-II, Beclin 1 and the protein level of p-p38 were significantly increased in the CSE-HBE cells. Compared to the CSE group, the different doses of XFPC medicated serum increased cell viability, decreased cell apoptosis, and inhibited mRNA and protein expression of LC3-I, LC3-II, Beclin 1 and protein level of p-p38. These results were especially observed in the group XFPC-H. After adding a p38 agonist, the therapeutic effect of XFPC on cell viability and autophagy was suppressed. Conclusion: XFPC significantly increased cell viability in a CSE-induced HBE cell model for chronic obstructive pulmonary disease through inhibiting the level of autophagy mediated by phosphorylation of p38.

Therapeutic effect of co-culture of rat bone marrow mesenchymal stem cells and degenerated nucleus pulposus cells on intervertebral disc degeneration

BackgroundAfter non-contact co-culture of bone marrow mesenchymal stem cells (BMSCs) with nucleus pulposus cells (NPCs), exosomes secreted by BMSCs were able to ameliorate the degree of disc degeneration. The reason for this is, at least in part, that exosomes from BMSCs achieve by affecting the level of autophagy in NPCs, while the components in exosomes are diverse and their specific mechanism of action is still unclear. PurposeHere, we aimed to explore the therapeutic effect of co-culture of BMSCs and NPCs on NPCs and explore its specific mechanism of action. Study design/SettingIn vitro study. MethodsRat NPCs and BMSCs were isolated and cultured in vitro. The serum deprivation experiment (using oxygen, glucose, and serum deprivation [OGD]) simulates the pathological state of low blood supply of the intervertebral disc in vivo. We used apoptotic cell staining and flow cytometry to study the effect of BMSCs on the apoptosis rate of rat NPCs, and the apoptotic proteins active-caspase-3, active-caspase-9, autophagy marker proteins LC3 and Beclin 1 were further detected using Western blot analysis. The expression levels of the pro-apoptotic protein Bax and the apoptosis-inhibiting protein Bcl2 were measured. The differentially expressed miRNAs were screened in a gene expression profiling chip. Then qRT-PCR was used to detect the effect of different treatment methods on miR-155 expression. The effect of anti-miR-155 antibodies on autophagy was studied by flow cytometry and transmission electron microscopy. A luciferase reporter assay was used to study the direct interaction between miR-155 and BACH1 mRNA, which was analyzed by TargetScan software, and the results were verified by Western blotting. ResultsCompared with the OGD group, the expression level of miR-155 and the NPC autophagy level significantly increased; the HO-1 protein expression increased; and the Bach1 protein expression, degeneration index, and apoptosis index all significantly decreased in the co-culture group. After BMSCs transfected with anti-miR-155 were co-cultured with NPCs, the miR-155 expression in the cells was significantly reduced, the HO-1 protein expression and the level of cell autophagy was reduced. However, Bach1 protein expression, NPC degeneration index, and apoptosis index increased. After being inhibited by the autophagy inhibitor wortmannin, the cell degeneration index and apoptosis rate significantly improved. ConclusionIn the OGD model, BMSCs can significantly increase the viability, the level of autophagy, and reduce the level of apoptosis in rat NPCs. BMSC exosomes increase miR-155 expression in NPCs, which targets Bach1 and in turn upregulates HO-1 expression, activates autophagy in NPCs, inhibits the apoptosis level, and improves intervertebral disc degeneration. Clinical SignificanceOur experiment shows that it is maybe feasible to treat disc degeneration with drugs. At the same time, compared with BMSC injection method of treatment, side effects of drug therapy are smaller, and can be controlled, it also provides a new way for intervertebral disc degeneration drug treatment.

LncRNA-HOTAIR activates autophagy and promotes the imatinib resistance of gastrointestinal stromal tumor cells through a mechanism involving the miR-130a/ATG2B pathway

Gastrointestinal stromal tumors (GISTs) are common neoplasms of the gastrointestinal tract that can be treated successfully using C-kit target therapy and surgery; however, imatinib chemoresistance is a major barrier to success in therapy. The present study aimed to discover alternative pathways in imatinib-resistant GISTs. Long noncoding RNAs (lncRNAs) are newly discovered regulators of chemoresistance. Previously, we showed that the lncRNA HOTAIR was upregulated in recurrent GISTs. In this study, we analyzed differentially expressed lncRNAs after imatinib treatment and found that HOTAIR displayed the largest increase. The distribution of HOTAIR in GISTs was shifted from nucleus to cytoplasm after imatinib treatments. The expression of HOTAIR was validated as related to drug sensitivity through Cell Counting Kit-8 assays. Moreover, HOTAIR was associated strongly with cell autophagy and regulated drug sensitivity via autophagy. Mechanistically, HOTAIR correlated negatively with miRNA-130a in GISTs. The downregulation of miRNA-130a reversed HOTAIR-small interfering RNA-induced suppression of autophagy and imatinib sensitivity. We identified autophagy-related protein 2 homolog B (ATG2B) as a downstream target of miR-130a and HOTAIR. ATG2B downregulation reversed the effect of pEX-3-HOTAIR/miR-130a inhibitor on imatinib sensitivity. Finally, HOTAIR was shown to influence the autophagy and imatinib sensitivity of GIST cells in mouse tumor models. Our results suggested that HOTAIR targets the ATG2B inhibitor miR-130a to upregulate the level of cell autophagy so that promotes the imatinib resistance in GISTs.

Astragaloside Ⅳ's Therapeutic Effect on Myocardial Infarction via Affecting Autophagy and the Mechanism Study

The purpose of this study was to investigate the protective effect of astragaloside Ⅳ (AS-Ⅳ) on neonatal rats' hypoxic/reoxygenated (H/R) injured myocardial cells and to explore its underlying mechanism. Cardiac cells were extracted from newborn rats and divided into control, H/R, H/R-low AS-Ⅳ (0.1 μmol/L AS-Ⅳ), H/R-medium AS-Ⅳ (1 μmol/L AS-Ⅳ), H/R-high AS-Ⅳ (10 μmol/L AS-Ⅳ) and H/R-high AS-Ⅳ-AKT (10 μmol/L AS-Ⅳ+5 μmol/L AKT) groups. After 48 h of treatment, the contents of LC3-Ⅱ, p62, AKT, pAKT, rapamycin (mTOR) mammalian targets and uncoordinated 51-like kinase 1 (ULK1) in cardiac myocytes were compared. Immunofluorescence staining was used to detect the expression of P62 in myocardium autophagosome. AS-Ⅳ improved the proliferative activity of cardio AS-Ⅳ improved the proliferative activity of cardiomyocytes in H/R injury in a dose-dependent manner and inhibited the level of cell autophagy. However, when AKT inhibitors were added, the effect of AS-Ⅳ was partially inhibited ( P<0.05). Gene and protein expression showed that AS-Ⅳ had no significant effect on the expression of AKT and mTOR genes ( P>0.05), but could significantly promote the phosphorylation of AKT and mTOR ( P<0.05). Immunofluorescence staining results showed that high concentrations of the AS - Ⅳ can reverse H/R injury induced the expression of autophagy body P62. AS-Ⅳ showed protection effect on H/R injured myocardial cells. The possible mechanism is by reducing the autophagy level via activating the mTOR signal in the PI3K/AKT pathway, thereby preventing H/R damage in neonatal rat cardiomyocytes.

Rapamycin Protects Skin Fibroblasts From UVA-Induced Photoaging by Inhibition of p53 and Phosphorylated HSP27.

Skin aging caused by UV radiation is called photoaging is characterized by skin roughness and dryness accompanied by a significant reduction of dermal collagen. Rapamycin is a macrolide immunosuppressant which has been shown to exhibit “anti-aging” effects in cells and organisms, however, its roles in the skin photoaging remains unclear. Here, we investigate the role of rapamycin and HSP27, which we have previously identified as an inhibitor of UV-induced apoptosis and senescence in HaCat cells, in a UVA-induced photoaging model of primary human dermal fibroblasts (HDFs). Results from senescence-associated beta-galactosidase (SA-β-gal) staining revealed that rapamycin significantly reduced senescence in UVA-treated HDFs. In addition, treatment with rapamycin significantly increased cell autophagy levels, decreased the expression of p53 and phosphorylated HSP27, and reduced genotoxic and oxidative cellular stress levels in UVA-induced HDFs. Knockdown of HSP27 resulted in a significant increase of MMP-1 and MMP-3 as well as a decrease in type I collagen expression. Rapamycin mitigated these effects by activation of the classical TGF-β/Smad signaling pathway and increasing the transcriptional activity of MAPK/AP-1. Taken together, these results suggest that rapamycin may potentially serve as a preventive and therapeutic agent for UVA-induced photoaging of the skin.

Intracellular alpha-fetoprotein interferes with all-trans retinoic acid induced ATG7 expression and autophagy in hepatocellular carcinoma cells

Retinoic acid and retinoid acid receptor (RA-RAR) signaling exhibits suppressive functions in the progression of hepatocellular carcinoma (HCC) through multiple mechanisms. However, whether RA-RAR signaling induces autophagy that contributes its anti-tumor activity in HCC remains elusive. In the current study, the effects of RA-RAR pathway on autophagy were investigated in two HCC cell lines: alpha-fetoprotein (AFP) positive PLC/PRF/5 and AFP negative HLE cells. Cell autophagy was analyzed with western blot for detection of LC3 conversion and p62/SQSTM1 degradation while autophagy flux was assayed using the mRFP-GFP-LC3 reporter. Cell apoptosis and viability were analyzed by caspase-3 activity, TdT-mediated dUTP nick end labeling (TUNEL) assay, and Cell Counting Kit (CCK)-8, respectively. Chromatin immunoprecipitation (ChIP) was employed to detect the binding of RAR onto the promoter of autophagy-relevant 7 (ATG7), and co-immunoprecipitation (CoIP) was used to analyze the interaction of AFP and RAR. The results showed that ATRA dosage and time-dependently induced high levels of cell autophagy in both the PLC/PRF/5 and HLE cells, which was accompanied with up-regulation of ATG7. ChIP assay showed that RAR was able to bind to its responsive elements on ATG7 promoter. Impairment of ATG7 induction or blockade of autophagy with chloroquine aggravated ATRA induced apoptosis of HCC cells. Furthermore, intracellular AFP was able to complex with RAR in PLC/PRF/5 cells. Knockdown of AFP in PLC/PRF/5 cells augmented the up-regulation of ATG7 by ATRA while overexpression of AFP in HLE cells attenuated ATRA induced ATG7 expression and autophagy. Thus, ATRA induced ATG7 and autophagy participated in its cytotoxicity on HCC cells and AFP interfere with the induction of ATG7 and autophagy through forming complex with RAR.

Biomarkers of Autophagy.

Biomarkers (short for biological markers) are biological measures of a biological state. Autophagy biomarkers play an important role as an indicator of autophagy during normal physiological processes, pathogenic processes or pharmacological responses to drugs. In this chapter, some biomarkers of different types of autophagy, including macroautophagy, selective autophagy, chaperone-mediated autophagy, and microautophagy, as well as the lysosomal biomarkers are introduced. The described biomarkers may be used to detect the level of autophagy in cells or tissues in a dynamic, real-time, and quantitative manner. However, each biomarker has its specific significance and limitation. Therefore, the analysis of the autophagy level in cells or tissues through the detection of autophagy biomarkers should be carried out carefully.

Curcumin prevents tension-induced endplate cartilage degeneration by enhancing autophagy

AimsIntermittent cyclic tension stimulation(ICMT) was shown to promote degeneration of endplate chondrocytes and induce autophagy. However, enhancing autophagy can alleviate degeneration partly. Studies have shown that curcumin can induce autophagy and protect chondrocytes, we speculated that regulation of autophagy by curcumin might be an effective method to improve the stress resistance of endplate cartilage. In this study, human cervical endplate cartilage specimens were collected, and expression of autophagy markers was detected and compared. Main methodsHuman cervical endplate chondrocytes were cultured to establish a tension-induced degeneration model, for which changes of functional metabolism and autophagy levels were detected under different tension loading conditions. Changes in functional metabolism of endplate chondrocytes were observed under high-intensity tension loading in the presence of inhibitors, inducers, and curcumin to regulate the autophagy level of cells. In addition, a rat model of lumbar instability was established to observe the degeneration of lumbar disc after curcumin administration. Key findingsThrough a series of experiments, we found that low-intensity tension stimulation can maintain a stable phenotype of endplate chondrocytes, but high-intensity tension stimulation has a negative effect. Moreover, with increasing tension intensity, the degree of degeneration of endplate chondrocytes was gradually aggravated and the level of autophagy increased. Besides, curcumin upregulated autophagy, inhibited apoptosis, and reduced phenotype loss of endplate chondrocytes induced by high-intensity tension loading, thereby relieving intervertebral disc degeneration induced by mechanical imbalance. SignificanceCurcumin mediated autophagy and enhanced the adaptability of endplate chondrocytes to high-intensity tension load, thereby relieving intervertebral disc degeneration.

Liraglutide ameliorates COCl2-induced oxidative stress and apoptosis in H9C2 cells via regulating cell autophagy.

Protective effects of liraglutide on H9C2 cells cultured using CoCl2 and its mechanism of action were investigated. Hypoxia model was established using CoCl2-treated H9C2 cells. With liraglutide as the treatment factor, apoptosis, changes in nitric oxide (NO) and reactive oxygen species (ROS) activity, mitochondrial membrane potential and change in cell autophagy level were detected via Hoechst staining, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) and western blotting (WB), respectively. Liraglutide ameliorated the CoCl2-induced decrease in H9C2 cell viability, the increases in cytotoxicity and percentage of apoptotic cells as well as oxidative stress in cells. Moreover, it stimulated the elevation of cell autophagy level. However, the protective effects of liraglutide on H9C2 cells were attenuated remarkably after adding the cell autophagy inhibitor. Liraglutide can ameliorate the CoCl2-induced oxidative stress and apoptosis in H9C2 cells via regulating cell autophagy.

LncRNA-MALAT1 influences myocardial infarction by regulating miR-30a/beclin-1 pathway.

Long non-coding ribonucleic acid-metastasis-associated lung adenocarcinoma transcript 1 (lncRNA-MALAT1) has been confirmed as a key factor involving in various physiological and pathological processes. The present study aims to investigate whether lncRNA-MALAT1 affects the process of myocardial infarction (MI) in rats by regulating the micro RNA (miR-30a)/Beclin-1 (BECN1) pathway. Twelve healthy male Sprague-Dawley rats were randomly selected and equally divided into sham-operation group and MI group. In MI group, a rat model of acute MI (AMI) was established by ligating the left anterior descending coronary artery. Rats in sham-operation group were set as the control. The messenger RNA (mRNA) expression levels of lncRNA-MALAT1, miR-30a, and BECN1 in the infarcted myocardial tissues were detected via real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). The rat myocardial cell line H9c2 was cultured in vitro and then transfected with vectors of lncRNA-MALAT1, miR-30a, and BECN1. After that, qRT-PCR was performed to measure mRNA levels of lncRNA-MALAT1, miR-30a, and BECN1 in H9c2 cells. The protein level of BECN1 in cells was determined via Western blotting (WB) assay. Expression levels of lncRNA-MALAT1 and BECN1 in the rat myocardium of MI group were up-regulated markedly (p<0.01), while miR-30a was down-regulated notably (p<0.01) compared with those in sham-operation group. After H9c2 cells were transfected with overexpression vectors of lncRNA-MALAT1 or miR-30a, expression levels of miR-30a (p<0.01) and BECN1 (p<0.01) were remarkably down-regulated, respectively. LncRNA-MALAT1 up-regulates the expression of BECN1 by binding to miR-30a, thereby increasing the level of cell autophagy after MI.