scholarly journals Long non-coding RNA MALAT1 enhances the apoptosis of cardiomyocytes through autophagy inhibition by regulating TSC2-mTOR signaling

2019 ◽  
Vol 52 (1) ◽  
Author(s):  
Hao Hu ◽  
Jiawei Wu ◽  
Xiaofan Yu ◽  
Junling Zhou ◽  
Hua Yu ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Noncoding Rna ◽  
Mtor Signaling ◽  
Cardiomyocyte Apoptosis ◽  
Tunel Staining ◽  
Promoter Regions ◽  
Related Proteins ◽  
Autophagy Inhibition

Abstract Background Our previous study showed that knockdown of long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) attenuated myocardial apoptosis in mouse acute myocardial infarction (AMI). This study aims to explore whether MALAT1 enhanced cardiomyocyte apoptosis via autophagy regulation and the underlying mechanisms of MALAT1 regulating autophagy. Methods Cardiomyocytes were isolated from neonatal mice and then stimulated with hypoxia/reoxygenation (H/R) injury to mimic AMI. The autophagy level was assessed using GFP-LC3 immunofluorescence and western blot analysis of autophagy-related proteins. RNA pull-down and RNA immunoprecipitation (RIP) was performed to analyze the binding of MALAT1 and EZH2. Chromatin immunoprecipitation (ChIP) assay was performed to analyze the binding of TSC2 promoter and EZH2. The cell apoptosis was evaluated using TUNEL staining and western blot analysis of apoptosis-related proteins. Results H/R injury increased MALAT1 expression in cardiomyocytes. Furthermore, MALAT1 overexpression inhibited, whereas MALAT1 knockdown enhanced the autophagy of cardiomyocytes. Moreover, MALAT1 overexpression recruited EZH2 to TSC2 promoter regions to elevate H3K27me3 and epigenetically inhibited TSC2 transcription. Importantly, TSC2 overexpression suppressed mTOR signaling and then activated the autophagy. Further results showed that MALAT1 inhibited proliferation and enhanced apoptosis of cardiomyocytes through inhibiting TSC2 and autophagy. Conclusion These findings demonstrate that the increased MALAT1 expression induced by H/R injury enhances cardiomyocyte apoptosis through autophagy inhibition by regulating TSC2-mTOR signaling.

Long noncoding RNA MALAT1 enhances the apoptosis of cardiomyocytes through autophagy modulation

2020 ◽  
Vol 98 (2) ◽  
pp. 130-136
Author(s):  
Hao Hu ◽  
Jiawei Wu ◽  
Xiaofan Yu ◽  
Junling Zhou ◽  
Hua Yu ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Long Noncoding Rna ◽  
Western Blot Analysis ◽  
Noncoding Rna ◽  
Cardiomyocyte Apoptosis ◽  
Tunel Staining ◽  
Myocardial Apoptosis ◽  
Related Proteins ◽  
Cardiomyocyte Survival

Induction of autophagy promotes cardiomyocyte survival and confers a cardioprotective effect on acute myocardial infarction (AMI). Our previous study showed that knockdown of long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) attenuated myocardial apoptosis in mouse AMI. Herein, this study further investigated whether the mechanisms by which MALAT1 enhanced cardiomyocyte apoptosis involved the autophagy regulation. To address this, cardiomyocytes were isolated from neonatal mice and then stimulated with hypoxia/reoxygenation (H/R) injury to mimic AMI. The cell apoptosis was evaluated using TUNEL staining and Western blot analysis of apoptosis-related proteins. The autophagy level was assessed using GFP-LC3 immunofluorescence and Western blot analysis of autophagy-related proteins. The results showed that H/R injury increased MALAT1 expression. Furthermore, MALAT1 overexpression significantly enhanced apoptosis and regulated autophagy of cardiomyocytes, whereas MALAT1 knockdown exerted the opposite effect. Moreover, rapamycin (an autophagy activator) effectively attenuated the MALAT1-mediated enhancement of cardiomyocyte apoptosis. Overall, our findings demonstrated that the increased MALAT1 expression induced by H/R injury enhances cardiomyocyte apoptosis, at least in part, through autophagy modulation.

scholarly journals Punicalagin Prevents Inflammation in LPS-Induced RAW264.7 Macrophages by Inhibiting FoxO3a/Autophagy Signaling Pathway

Nutrients ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 2794 ◽  
Author(s):  
Cao ◽  
Chen ◽  
Ren ◽  
Zhang ◽  
Tan ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Inflammatory Cytokines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Inflammatory Responses ◽  
Raw264.7 Macrophages ◽  
Tnf Α ◽  
Autophagy Inhibition ◽  
Pro Inflammatory Cytokines

Punicalagin, a hydrolysable tannin of pomegranate juice, exhibits multiple biological effects, including inhibiting production of pro-inflammatory cytokines in macrophages. Autophagy, an intracellular self-digestion process, has been recently shown to regulate inflammatory responses. In this study, we investigated the anti-inflammatory potential of punicalagin in lipopolysaccharide (LPS) induced RAW264.7 macrophages and uncovered the underlying mechanisms. Punicalagin significantly attenuated, in a concentration-dependent manner, LPS-induced release of NO and decreased pro-inflammatory cytokines TNF-α and IL-6 release at the highest concentration. We found that punicalagin inhibited NF-κB and MAPK activation in LPS-induced RAW264.7 macrophages. Western blot analysis revealed that punicalagin pre-treatment enhanced LC3II, p62 expression, and decreased Beclin1 expression in LPS-induced macrophages. MDC assays were used to determine the autophagic process and the results worked in concert with Western blot analysis. In addition, our observations indicated that LPS-induced releases of NO, TNF-α, and IL-6 were attenuated by treatment with autophagy inhibitor chloroquine, suggesting that autophagy inhibition participated in anti-inflammatory effect. We also found that punicalagin downregulated FoxO3a expression, resulting in autophagy inhibition. Overall these results suggested that punicalagin played an important role in the attenuation of LPS-induced inflammatory responses in RAW264.7 macrophages and that the mechanisms involved downregulation of the FoxO3a/autophagy signaling pathway.

scholarly journals Endoplasmic Reticulum Stress is Involved in DFMO Attenuating Isoproterenol-Induced Cardiac Hypertrophy in Rats

2016 ◽  
Vol 38 (4) ◽  
pp. 1553-1562 ◽  
Author(s):  
Yan Lin ◽  
Xiaojie Zhang ◽  
Wei Xiao ◽  
Bo Li ◽  
Jun Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endoplasmic Reticulum ◽  
Endoplasmic Reticulum Stress ◽  
Cardiac Hypertrophy ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cardiac Fibrosis ◽  
Regulation Of Gene Expression ◽  
Tunel Staining

Background/Aims: Studies performed in experimental animals have shown that polyamines contribute to several physiological and pathological processes, including cardiac hypertrophy. This involves an increase in ornithine decarboxylase (ODC) activity and intracellular polyamines associated with regulation of gene expression. Difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, has attracted considerable interest for its antiproliferative role, which it exerts through inhibition of the polyamine pathway and cell turnover. Whether DFMO attenuates cardiac hypertrophy through endoplasmic reticulum stress (ERS) is unclear. Methods: Myocardial hypertrophy was simulated by isoproterenol (ISO). Polyamine depletion was achieved using DFMO. Hypertrophy was estimated using the heart/body index and atrial natriuretic peptide (ANP) gene expression. Cardiac fibrosis and apoptosis were measured by Masson and TUNEL staining. Expression of ODC and spermidine/spermine N1-acetyltransferase (SSAT) were analyzed via real-time PCR and Western blot analysis. Protein expression of ERS and apoptosis factors were analyzed using Western blot analysis. Results: DFMO treatments significantly attenuated hypertrophy and apoptosis induced by ISO in cardiomyocytes. DFMO down-regulated the expression of ODC, glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), cleaved caspase-12, and Bax and up-regulated the expression of SSAT and Bcl-2. Finally, these changes were partially reversed by the addition of exogenous putrescine. Conclusion: The data presented here suggest that polyamine depletion could inhibit cardiac hypertrophy and apoptosis, which is closely related to the ERS pathway.

scholarly journals The role of activating transcription factor 6 in hydroxycamptothecin-induced fibroblast autophagy and apoptosis

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Jin Tao ◽  
Hui Chen ◽  
Xiaolei Li ◽  
Jingcheng Wang
Keyword(s):  
Transcription Factor ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cell Counting ◽  
Immunofluorescent Staining ◽  
Fibroblast Proliferation ◽  
Activating Transcription Factor ◽  
Related Proteins

Abstract Background The over-proliferation of fibroblasts is considered to be the main cause of scar adhesion after joint surgery. Hydroxycamptothecin (HCPT), though as a potent antineoplastic drug, shows preventive effects on scar adhesion. This study aimed to investigate the role of activating transcription factor 6 (ATF-6) in the HCPT-induced inhibition of fibroblast viability. Methods The cell counting kit-8 (CCK-8) assay, western blot analysis, lentivirus-mediated gene silencing, transmission electron microscopy (TEM) analysis, immunofluorescent staining for autophagy-related protein light chain 3 (LC3) were used to explore the effect of HCPT on triggering fibroblast apoptosis and inhibiting fibroblast proliferation, and the involvement of possible signaling pathways. Results It was found that HCPT exacerbated fibroblast apoptosis and repressed its proliferation. Subsequently, endoplasmic reticulum stress (ERS)-related proteins were determined by western blot prior to ATF6 p50 was screened out and reexamined after it was silenced. As a result, ATF6-mediated ERS played a role in HCPT-induced fibroblast apoptosis. Autophagy-related proteins and autophagosomes were detected after the HCPT administration using western blot and TEM analyses, respectively. Autophagy was activated after the HCPT treatment. With the co-treatment of autophagy inhibitor 3-methyladenine (3-MA), both the western blot analysis and the CCK-8 assay showed inhibited autophagy, which indicated that the effect of HCPT on fibroblast proliferation was partially reversed. Besides, the LC3 immunofluorescence staining revealed suppressed autophagy after silencing ATF6 p50. Conclusion Our results demonstrate that HCPT acts as a facilitator of fibroblast apoptosis and inhibitor of fibroblast proliferation for curbing the postoperative scar adhesion, in which the ATF6-mediated ERS pathway and autophagy are involved.

scholarly journals Critical Roles of the PI3K-Akt-mTOR Signaling Pathway in Apoptosis and Autophagy of Astrocytes Induced by Methamphetamine

2019 ◽  
Vol 17 (1) ◽  
pp. 96-104 ◽  
Author(s):  
Han-Qing Liu ◽  
Ya-Wen An ◽  
A-Zhen Hu ◽  
Ming-Hua Li ◽  
Jue-Lian Wu ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Critical Role ◽  
Mammalian Target Of Rapamycin ◽  
Mtor Signaling ◽  
Cell Counting ◽  
Mtor Signaling Pathway ◽  
Induced Apoptosis

AbstractThis study aimed to reveal potential roles of the phosphatidylinositol 3 kinase (PI3K)-protein kinase B (Akt)-mammalian target of rapamycin (mTOR) signaling pathway in apoptosis and autophagy of astrocytes induced by methamphetamine (METH). A Cell Counting Kit-8 (CCK-8) was used to determine the reduction in proliferation of U-118 MG cells induced by METH. Hoechst 33258 and flow cytometry were used to observe the astrocytes. Western blot analysis was performed to evaluate protein expression and phosphorylation levels. METH inhibited the proliferation of U-118 MG cells and induced apoptosis and autophagy. Western blot analysis showed that the ratio of LC3-II/I was increased, whereas the expression of Bcl-2 was decreased. The phosphorylation cascade of kinases in the PI3K-Akt-mTOR signaling pathway was significantly inhibited by METH exposure, as were proteins downstream of mTORC1, such as p70s6k, rps6, 4EBP1 and eIF4E. METH inhibited proliferation of U-118 MG cells and induced apoptosis and autophagy. The PI3K-Akt-mTOR signaling pathway likely plays a critical role in these effects.

scholarly journals Zeaxanthin Exhibits Protective Effects in Myocardial Injury by Inhibiting TGF-β/Smad2/3 and p38MAPK/NF-κB Signaling Pathways

2021 ◽  
Author(s):  
Mingyang Li ◽  
Xiang Song ◽  
Lichun Qi ◽  
Yanhui Gao ◽  
Xin Wang ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathways ◽  
Signaling Pathway ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Myocardial Injury ◽  
Transforming Growth Factor ◽  
Cardiomyocyte Apoptosis ◽  
Related Protein ◽  
Tgf Β1

Abstract Background: Zeaxanthin is a newly discovered natural product in β-carotenoid family with multiple bioactivities. Recently, it has been shown that zeaxanthin may have cardioprotective effects in several studies, but its mechanisms have not been fully investigated. Herein, we explored the role and mechanism of zeaxanthin in myocardial injury.Methods and Results: In this study, three different models were used to investigate the mechanism by which zeaxanthin alleviates myocardial injury. H9C2 Cardiomyocyte injury models were induced by H2O2. TUNEL assay, Flow cytometry, and Western blot analysis showed that treatment with zeaxanthin significantly decreased cardiomyocyte apoptosis and apoptosis-related protein expression. And reactive oxygen species (ROS) measurement analysis and Western blot analysis showed that treatment with zeaxanthin also could reduce the production of ROS and affect the expression of p38-Mitogen activated protein kinase/nuclear factor-κ gene bindin (p38MAPK/NF-κB) signaling pathway. Transforming Growth Factor-β1 (TGF-β1) was used to establish the fibrosis model in cardiac fibroblasts (CFs). QRT-PCR and Western blot analysis showed that treatment with zeaxanthin significantly decreased the expression of fibrosis markers in CFs. Myocardial injury animal models were induced by high-fat diet (HFD). Our results demonstrated that zeaxanthin improved fibrosis damage and cardiomyocyte apoptosis in HFD mice. Furthermore, Western blot analysis showed that TGF-β/Drosophila mothers against decapentaplegic2/3 (TGF-β/Smad2/3) signaling pathway related protein p-Smad2/3, Smad2/3, and TGF-β1 were significantly downregulated by zeaxanthin treatment.Conclusions: Zeaxanthin may alleviate HFD and H2O2-induced heart injury by regulating TGF-β/Smad2/3 and p38MAPK/NF-κB signaling pathways, which is of immense clinical significance in the treatment of cardiovascular disease.

scholarly journals Microvesicles Derived from Human Embryonic Neural Stem Cells Inhibit the Apoptosis of HL-1 Cardiomyocytes by Promoting Autophagy and Regulating AKT and mTOR via Transporting HSP-70

2019 ◽  
Vol 2019 ◽  
pp. 1-15 ◽  
Author(s):  
Lei Zhang ◽  
Jianyi Gao ◽  
Tianyan Chen ◽  
Xiang Chen ◽  
Xianyan Ji ◽  
...  
Keyword(s):  
Stem Cells ◽  
Heat Shock ◽  
Neural Stem Cells ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Signalling Pathway ◽  
Hsp 70 ◽  
Related Proteins ◽  
Mtor Signalling

Myocardial reperfusion injury (MRI) induced by cardiomyocyte apoptosis plays an important role in the pathogenesis of a variety of cardiovascular diseases. New MRI treatments involving stem cells are currently being developed because these cells may exert their therapeutic effects primarily through paracrine mechanisms. Microvesicles (MVs) are small extracellular vesicles that have become the key mediators of intercellular communication. MVs derived from stem cells have been reported to play an important role in MRI. In this article, we attempted to explore the mechanisms by which MVs derived from human embryonic neural stem cells (hESC-NSC-derived MVs) rescue MRI. hESCs were differentiated into NSCs, and MVs were isolated from their supernatants by ultracentrifugation. H2O2 was used to induce apoptosis in HL-1 cardiomyocytes. Cell viability was detected by using the CCK-8 assay, apoptosis was detected by Annexin V-FITC/PI staining, and apoptosis-related proteins and signalling pathway-related proteins were detected by western blot analysis. Autophagic flux was measured using the tandem fluorescent mRFG-GFP-LC3 assay. Transmission electron microscopy and western blot analysis were adopted to evaluate autophagy levels. hESC-NSC-derived MVs increased the autophagy and inhibited the apoptosis of HL-1 cells exposed to H2O2 for 3 h in a dose-dependent manner. Additionally, hESC-NSC-derived MVs contained high levels of heat shock protein 70 (HSP-70), which can increase the level of HSP-70 in cells. Moreover, the same effect could be achieved by heat shock preconditioning of HL-1 cells overexpressing HSP-70. The benefits of NSC-MVs may be due to the involvement of AKT and mTOR signalling pathways. Importantly, hESC-NSC-derived MVs stimulated the activation of the AKTand mTOR signalling pathway in those cells by transporting HSP-70. Our results suggest that hESC-NSC-derived MVs inhibit the apoptosis of HL-1 cardiomyocytes by promoting autophagy and regulating AKT and mTOR via transporting HSP-70. However, this hypothesis requires in vivo confirmation.

scholarly journals Silencing the Expression of Cyclin G1 Enhances the Radiosensitivity of Hepatocellular Carcinoma in vitro and in vivo by Inducing Apoptosis

2020 ◽  
Author(s):  
Gang Xu ◽  
Shanshan Bu ◽  
Xiushen Wang ◽  
Hong Ge
Keyword(s):  
Hepatocellular Carcinoma ◽  
Western Blot ◽  
Blot Analysis ◽  
Hepg2 Cells ◽  
Western Blot Analysis ◽  
Cyclin G1 ◽  
Related Proteins ◽  
Hcc Cells

Abstract Background: Radiotherapy plays an important role in the treatment of hepatocellular carcinoma (HCC). Cyclin G1 was a novel member of the cyclin family, and it is abnormally expressed in HCC. The aim of this study was to investigate the role of cyclin G1 in the radiotherapy of HCC cells. Methods: The expression of cyclin G1 was silenced by transfection of cyclin G1-siRNA into HepG2 cells, and the expression of cyclin G1 mRNA and protein was measured by qRT-PCR and western blot analysis. The proliferation was analysed by MTT assay, and the radiosensitivity of HCC cells was detected by using a colony formation assay and a xenograft tumour model. The expression of apoptosis-related proteins (Bcl-2 and Bax) was detected by western blot analysis. Results: The expression of cyclin G1 mRNA and protein in HepG2-cyclin G1-siRNA cells is significantly decreased compared with that in HepG2 cells. Silencing the expression of cyclin G1 inhibits the proliferation of HCC cells and enhances the radiosensitivity of HepG2 cells in vitro and in vivo. HepG2-cyclin G1-siRNA significantly decreases Bcl-2 expression and increases Bax expression in cells.Conclusion: Silencing the expression of cyclin G1 enhances the radiosensitivity of HCC cells in vitro and in vivo, and the mechanism is probably related to the regulation of apoptosis-related proteins.

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