Endoplasmic reticulum stress regulates cell injury in lipopolysaccharide-induced nerve cells

Objective Sepsis-associated encephalopathy (SAE) is a common complication of sepsis, and excessive endoplasmic reticulum (ER) stress is closely correlated with the cell injury caused by sepsis. This study aimed to analyze the possible role of ER stress in SAE cell models. Methods PC12 and MES23.5 cells were treated with increasing concentrations of lipopolysaccharides (LPS). The Cell Counting Kit-8 assay was used to detect cell viability and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed to assess cell apoptosis. In addition, the protein expression levels of ER stress markers [GRP78, CHOP, inositol-requiring enzyme 1 (IRE1), and PKR-like ER kinase (PERK)] and apoptosis-related proteins (Bax, Bcl-2, caspase-3, and cleaved caspase-3) were analyzed using western blotting. Results LPS treatment activated ER stress markers in both the PC12 and MES23.5 cells. The overexpression of GRP78 significantly reduced cell viability and enhanced cell apoptosis in a time-dependent manner. An ER stress inhibitor, 4-PBA, significantly enhanced cell viability and inhibited the cell apoptosis induced by LPS. Therefore, an enhanced unfolded protein response (UPR) and UPR suppression may regulate cell apoptosis. Conclusions UPR was shown to be involved in regulating LPS-induced neuron injury. UPR could be a potential therapeutic target in SAE.

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EGFRvIII Promotes Cell Survival during Endoplasmic Reticulum Stress through a Reticulocalbin 1-Dependent Mechanism

Cancers ◽

10.3390/cancers13061198 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1198

Author(s):

Juliana Gomez ◽

Zammam Areeb ◽

Sarah F. Stuart ◽

Hong P. T. Nguyen ◽

Lucia Paradiso ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Cell Viability ◽

Er Stress ◽

Cell Survival ◽

Glioblastoma Cells ◽

Over Expression ◽

Stress Markers ◽

Apoptotic Protein ◽

Novel Association ◽

Reduced Activity

Reticulocalbin 1 (RCN1) is an endoplasmic reticulum (ER)-residing protein, involved in promoting cell survival during pathophysiological conditions that lead to ER stress. However, the key upstream receptor tyrosine kinase that regulates RCN1 expression and its potential role in cell survival in the glioblastoma setting have not been determined. Here, we demonstrate that RCN1 expression significantly correlates with poor glioblastoma patient survival. We also demonstrate that glioblastoma cells with expression of EGFRvIII receptor also have high RCN1 expression. Over-expression of wildtype EGFR also correlated with high RCN1 expression, suggesting that EGFR and EGFRvIII regulate RCN1 expression. Importantly, cells that expressed EGFRvIII and subsequently showed high RCN1 expression displayed greater cell viability under ER stress compared to EGFRvIII negative glioblastoma cells. Consistently, we also demonstrated that RCN1 knockdown reduced cell viability and exogenous introduction of RCN1 enhanced cell viability following induction of ER stress. Mechanistically, we demonstrate that the EGFRvIII-RCN1-driven increase in cell survival is due to the inactivation of the ER stress markers ATF4 and ATF6, maintained expression of the anti-apoptotic protein Bcl-2 and reduced activity of caspase 3/7. Our current findings identify that EGFRvIII regulates RCN1 expression and that this novel association promotes cell survival in glioblastoma cells during ER stress.

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MiR-140-3p inhibits the cell viability and promotes apoptosis of synovial fibroblasts in rheumatoid arthritis through targeting sirtuin 3

Journal of Orthopaedic Surgery and Research ◽

10.1186/s13018-021-02236-5 ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Beibei Zu ◽

Lin Liu ◽

Jingya Wang ◽

Meirong Li ◽

Junxia Yang

Keyword(s):

Rheumatoid Arthritis ◽

Cell Viability ◽

Cell Apoptosis ◽

Caspase 3 ◽

Pearson Correlation ◽

Fibrous Tissue ◽

Synovial Fibroblasts ◽

Cell Counting ◽

Sirtuin 3 ◽

Fibrous Tissues

Abstract Background Synovial fibroblasts (SFs) with the abnormal expressions of miRNAs are the key regulator in rheumatoid arthritis (RA). Low-expressed miR-140-3p was found in RA tissues. Therefore, we attempted to investigate the effect of miR-140-3p on SFs of RA. Methods RA and normal synovial fibrous tissue were gathered. The targets of miR-140-3p were found by bioinformatics and luciferase analysis. Correlation between the expressions of miR-140-3p with sirtuin 3 (SIRT3) was analyzed by Pearson correlation analysis. After transfection, cell viability and apoptosis were detected by cell counting kit-8 and flow cytometry. The expressions of miR-140-3p, SIRT3, Ki67, Bcl-2, Bax, and cleaved Caspase-3 were detected by RT-qPCR or western blot. Results Low expression of miR-140-3p and high expression of SIRT3 were found in RA synovial fibrous tissues. SIRT3 was a target of miR-140-3p. SIRT3 expression was negatively correlated to the expression of miR-140-3p. MiR-140-3p mimic inhibited the MH7A cell viability and the expressions of SIRT3, Ki67, and Bcl-2 and promoted the cell apoptosis and the expressions of Bax and cleaved Caspase-3; miR-140-3p inhibitor showed an opposite effect to miR-140-3p mimic on MH7A cells. SIRT3 overexpression not only promoted the cell viability and inhibited cell apoptosis of MH7A cells but also reversed the effect of miR-140-3p mimic had on MH7A cells. Conclusions The results in this study revealed that miR-140-3p could inhibit cell viability and promote apoptosis of SFs in RA through targeting SIRT3.

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Abstract 276: SR-BI Suppresses Apoptosis by Reducing Endoplasmic Reticulum Stress and Promoting Akt Activity in Macrophages

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.34.suppl_1.276 ◽

2014 ◽

Vol 34 (suppl_1) ◽

Author(s):

Huan Tao ◽

Patricia G Yancey ◽

Sean S Davies ◽

L Jackson Roberts ◽

John L Blakemore ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Apolipoprotein E ◽

Er Stress ◽

Cell Apoptosis ◽

Free Cholesterol ◽

Oxidized Ldl ◽

Tunel Staining ◽

Type I ◽

Apoptotic Cells ◽

Atherosclerotic Lesions

Objective: Macrophage apoptosis contributes to atherosclerotic plaque necrosis, inflammation, development and rupture. Scavenger receptor class B type I (SR-BI) is a key regulator of HDL metabolism and cellular cholesterol homeostasis. Here we examined the hypothesis that macrophage SR-BI modulates lipid-associated cellular stress and apoptosis. Methods and Results: In vitro cell apoptosis assays were performed in primary macrophages, and for in vivo evidence, we examined TUNEL staining of atherosclerotic lesions of LDLR -/- mice that were reconstituted with SR-BI -/- or WT bone marrow after 16 weeks on a Western diet. We found that SR-BI deficiency led to ~64.3% more apoptotic cells induced by oxidized LDL or free cholesterol in primary macrophages, and 6-fold more lesional apoptotic cells in SR-BI -/- →LDLR -/- mice compared to WT recipient mice. In macrophages, SR-BI deficiency caused significant accumulations of cellular free cholesterol and elevated markers of endoplasmic reticulum (ER) stress. These were exacerbated by feeding mice a high-cholesterol diet or inactivating the apolipoprotein E gene. Peroxidation of lipoproteins and cell membranes leads to modification of phosphatidylethanolamine by lipid aldehydes including isolevuglandins (IsoLG-PE). Treatment of macrophages with IsoLG-PE induced 52.6% more apoptotic cells in SR-BI -/- macrophages compared to WT. Transgenic expression of SR-BI by transfection of SR-BI -/- macrophages rescued oxidative stress-induced ER stress and cell apoptosis. SR-BI deficiency inhibited the Akt pathway compromising macrophage survival and increasing lesion necrosis. Moreover, Akt Activator was able to rescue SR-BI deficiency associated apoptosis in macrophages. Apolipoprotein E interacts with SR-BI in macrophages, co-operating for cellular lipid homeostasis and cell survival signaling. Conclusion: SR-BI protects against cell apoptosis induced by lipid stress in macrophages and atherosclerotic lesions. The underlying mechanisms are, at least in part, through reducing lipid-associated ER stress and promoting Akt activity in macrophages. Thus, we identify macrophage SR-BI-mediated apoptosis pathways as molecular targets for the prevention of atherosclerotic cardiovascular events.

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Endoplasmic Reticulum Stress Is Involved in Baicalin Protection on Chondrocytes From Patients With Osteoarthritis

Dose-Response ◽

10.1177/1559325818810636 ◽

2018 ◽

Vol 16 (4) ◽

pp. 155932581881063 ◽

Cited By ~ 1

Author(s):

Jiangang Cao ◽

Yu Zhang ◽

Tianyi Wang ◽

Bo Li

Keyword(s):

Oxidative Stress ◽

Endoplasmic Reticulum ◽

Endoplasmic Reticulum Stress ◽

Cell Viability ◽

Er Stress ◽

Messenger Rna ◽

Cell Counting ◽

Protective Effects ◽

Gene Expressions ◽

Protein Levels

Osteoarthritis (OA) affects elderly population worldwide and endoplasmic reticulum (ER) stress is known to be positively correlated with OA development. Previous reports prove the cytoprotective effects of baicalin on chondrocytes, whereas the mechanisms are hardly reported. Hence, we aimed to investigate the links between OA, ER stress, and baicalin. Chondrocytes from patients with OA were subjected to H2O2 treatment with or without baicalin pretreatment, and cell viability was assessed via Cell Counting Kit-8. Messenger RNA (mRNA) amounts of apoptosis-related genes (Bax, Bcl-2, and Caspase-3), extracellular matrix (ECM)-related genes (Collange I, Collange II, Aggrecan, and Sox9) and ER stress hallmarks (binding immunoglobulin protein [BiP] C/EBP homologous protein [CHOP]) were evaluated via quantitative real-time PCR. Bax, Bcl-2, BiP, and CHOP protein levels were analyzed via Western blot. Baicalin suppressed the changes in cell viability and apoptosis-related gene expressions caused by H2O2. Reactive oxygen species and glutathione/oxidized glutathione assay showed that H2O2 enhanced oxidative stress. Baicalin suppressed H2O2-induced downregulation of mRNA expression of ECM-related genes. Moreover, baicalin reduced H2O2-stimulated increase in oxidative stress and the expression of ER stress hallmarks. Endoplasmic reticulum stress inducer abolished the protective activities, whereas ER stress inhibitor did not exhibit extra protective effects. Baicalin pretreatment protected patient-derived chondrocytes from H2O2 through ER stress inhibition.

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Adiponectin Protects against Glutamate-Induced Excitotoxicity via Activating SIRT1-Dependent PGC-1αExpression in HT22 Hippocampal Neurons

Oxidative Medicine and Cellular Longevity ◽

10.1155/2016/2957354 ◽

2016 ◽

Vol 2016 ◽

pp. 1-12 ◽

Cited By ~ 19

Author(s):

Liang Yue ◽

Lei Zhao ◽

Haixiao Liu ◽

Xia Li ◽

Bodong Wang ◽

...

Keyword(s):

Western Blot ◽

Cell Viability ◽

Cell Apoptosis ◽

Hippocampal Neurons ◽

Caspase 3 ◽

Critical Role ◽

Tunel Staining ◽

Peroxisome Proliferator ◽

Protective Effects ◽

Expression Levels

Glutamate- (Glu-) induced excitotoxicity plays a critical role in stroke. This study aimed to investigate the effects of APN on Glu-induced injury in HT22 neurons. HT22 neurons were treated with Glu in the absence or the presence of an APN peptide. Cell viability was assessed using the MTT assay, while cell apoptosis was evaluated using TUNEL staining. Levels of LDH, MDA, SOD, and GSH-Px were detected using the respective kits, and ROS levels were detected using dichlorofluorescein diacetate. Western blot was used to detect the expression levels of silent information regulator 1 (SIRT1), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), cleaved caspase-3, Bax, and Bcl-2. In addition to the western blot, immunofluorescence was used to investigate the expression levels of SIRT1 and PGC-1α. Our results suggest that APN peptide increased cell viability, SOD, and GSH-Px levels and decreased LDH release, ROS and MDA levels, and cell apoptosis. APN peptide upregulated the expression of SIRT1, PGC-1α, and Bcl-2 and downregulated the expression of cleaved caspase-3 and Bax. Furthermore, the protective effects of the APN peptide were abolished by SIRT1 siRNA. Our findings suggest that APN peptide protects HT22 neurons against Glu-induced injury by inhibiting neuronal apoptosis and activating SIRT1-dependent PGC-1αsignaling.

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Bafilomycin Prevented Curcumin-Induced Endoplasmic Reticulum (ER) Stress and Autophagy in MCF-7 Growth Hormone Positive (GH+) Breast Cancer Cells

Proceedings ◽

10.3390/proceedings2251568 ◽

2018 ◽

Vol 2 (25) ◽

pp. 1568

Author(s):

Merve Karataş ◽

Ajda Coker-Gurkan ◽

Elif Damla Arisan ◽

Pınar Obakan-Yerlikaya ◽

Narcin Palavan-Unsal

Keyword(s):

Breast Cancer ◽

Growth Hormone ◽

Endoplasmic Reticulum ◽

Cell Viability ◽

Er Stress ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Time Dependent ◽

Dependent Manner ◽

Mcf 7

Autocrine growth hormone (GH) induced cell proliferation, invasion-metastasis and drug resistance in breast cancer cells. Curcumin has an apoptotic effect on colon, melanoma, cervix, and breast cancer cells. Autophagy and endoplasmic reticulum (ER) stress are essential cellular processes activated under nutrient deprivation, pathogen infection and drug exposure. Our aim in this study is to investigate the time-dependent effect of curcumin on ER stress and autophagy and potential increase of curcumin efficiency by bafilomycin treatment. Autocrine GH expression triggered resistant profile against curcumin-induced cell viability loss in MCF-7 cells. However, this effect was prevented by the time-dependent manner in MCF-7 cells. In GH+ breast cancer cells bafilomycin increase curcumin-induced cell viability loss by MTT cell viability assay. In conclusion, autocrine GH-triggered curcumin resistance was overcome by autophagy inhibition condition by bafilomycin treatment in a dose-dependent manner in MCF-7 GH+ breast cancer cells.

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A novel secretagogin/ATF4 pathway is involved in oxidized LDL-induced endoplasmic reticulum stress and islet β-cell apoptosis

Acta Biochimica et Biophysica Sinica ◽

10.1093/abbs/gmaa142 ◽

2020 ◽

Author(s):

Li Wu ◽

Yuncheng Lv ◽

Ying Lv ◽

Sunmin Xiang ◽

Zhibo Zhao ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Transcription Factor ◽

Er Stress ◽

Cell Apoptosis ◽

Initiation Factor ◽

Β Cells ◽

Β Cell ◽

Immunoprecipitation Assay ◽

Min6 Cells ◽

Stress Markers

Abstract Excessive accumulation of cholesterol in β cells initiates endoplasmic reticulum (ER) stress and associated apoptosis. We have reported that excessive uptake of cholesterol by MIN6 cells decreases the expression of secretagogin (SCGN) and then attenuates insulin secretion. Here, we aimed to determine whether cholesterol-induced SCGN decrease is involved in the modulation of ER stress and apoptosis in pancreatic β cells. In this study, MIN6 cells were treated with oxidized low-density lipoprotein (ox-LDL) for 24 h, and then intracellular lipid droplets and cell apoptosis were quantified, and SCGN and ER stress markers were identified by western blot analysis. Furthermore, small interfer RNA (siRNA)-mediated SCGN knockdown and recombinant plasmid-mediated SCGN restoration experiments were performed to confirm the role of SCGN in ER stress and associated cell apoptosis. Finally, the interaction of SCGN with ATF4 was computationally predicted and then validated by a co-immunoprecipitation assay. We found that ox-LDL treatment increased the levels of ER stress markers, such as phosphorylated protein kinase-like endoplasmic reticulum kinase, phosphorylated eukaryotic initiation factor 2 alpha, activating transcription factor 4 (ATF4), and transcription factor CCAAT-enhancer-binding protein homologous protein, and promoted MIN6 cell apoptosis; in addition, the expression of SCGN was downregulated. siRNA-mediated SCGN knockdown exacerbated β-cell ER stress by increasing ATF4 expression. Pretreatment of MIN6 cells with the recombinant SCGN partly antagonized ox-LDL-induced ER stress and apoptosis. Furthermore, a co-immunoprecipitation assay revealed an interaction between SCGN and ATF4 in MIN6 cells. Taken together, these results demonstrated that pancreatic β-cell apoptosis induced by ox-LDL treatment can be attributed, in part, to an SCGN/ATF4-dependent ER stress response.

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Dentatin Induces Apoptosis and Cell Cycle Arrest of Acute Myeloid Leukemia Cells

Current Topics in Nutraceutical Research ◽

10.37290/ctnr2641-452x.19:52-57 ◽

2020 ◽

Vol 19 (1) ◽

pp. 52-57

Author(s):

Li Wen ◽

Yuli Liang ◽

Jing Li ◽

Meijie Quan ◽

Yanxiao Li ◽

...

Keyword(s):

Cell Cycle ◽

Acute Myeloid Leukemia ◽

Cell Viability ◽

Cell Apoptosis ◽

Myeloid Leukemia ◽

Cell Counting ◽

Dependent Manner ◽

Cycle Arrest ◽

Acute Myeloid Leukemia Cells ◽

Acute Myeloid

Acute myeloid leukemia remains a therapeutic challenge in the medical field and improvement in chemotherapeutics is needed. In this paper, MOLM-13 cells were treated with different concentrations (0, 10, 50, 100 µM) of dentatin and cell viability was detected using Cell Counting Kit-8. Cell cycle and cell apoptosis rates were evaluated by flow cytometry. The relevant proteins were assessed by Western blot. Consequently, the results show that dentatin inhibits the cell viability in a dose-dependent manner. In addition, dentatin arrests the cell cycle at G1 phase (P ‹ 0.01). Moreover, dentatin induces the cell apoptosis. Further study revealed that dentatin downregulates the phosphorylated STAT3 and CyclinD1 but upregulates the cleaved caspase-3. In summary, this study confirms that dentatin inhibits MOLM-13 cell viability, increases cell apoptosis, and retards cell cycle.

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Endoplasmic Reticulum Stress Cooperates in Silica Nanoparticles-Induced Macrophage Apoptosis via Activation of CHOP-Mediated Apoptotic Signaling Pathway

International Journal of Molecular Sciences ◽

10.3390/ijms20235846 ◽

2019 ◽

Vol 20 (23) ◽

pp. 5846 ◽

Cited By ~ 6

Author(s):

Fenglei Chen ◽

Jiaqi Jin ◽

Jiahui Hu ◽

Yujing Wang ◽

Zhiyu Ma ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Signaling Pathway ◽

Cell Apoptosis ◽

Caspase 3 ◽

Raw 264.7 ◽

Apoptotic Signaling ◽

Macrophage Cells ◽

Raw 264.7 Macrophage ◽

Raw 264.7 Macrophage Cells

While silica nanoparticles (SiNPs) have wide applications, they inevitably increase atmospheric particulate matter and human exposure to this nanomaterial. Numerous studies have focused on how to disclose SiNP toxicity and on understanding its toxic mechanisms. However, there are few studies in the literature reporting the interaction between endoplasmic reticulum (ER) stress and SiNP exposure, and the corresponding detailed mechanisms have not been clearly determined. In this study, CCK-8 and flow cytometry assays demonstrated that SiNPs gradually decreased cell viability and increased cell apoptosis in RAW 264.7 macrophage cells in dose- and time-dependent manners. Western blot analysis showed that SiNPs significantly activated ER stress by upregulating GRP78, CHOP, and ERO1α expression. Meanwhile, western blot analysis also showed that SiNPs activated the mitochondrial-mediated apoptotic signaling pathway by upregulating BAD and Caspase-3, and downregulating the BCL-2/BAX ratio. Moreover, 4-phenylbutyrate (4-PBA), an ER stress inhibitor, significantly decreased GRP78, CHOP, and ERO1α expression, and inhibited cell apoptosis in RAW 264.7 macrophage cells. Furthermore, overexpression of CHOP significantly enhanced cell apoptosis, while knockdown of CHOP significantly protected RAW 264.7 macrophage cells from apoptosis induced by SiNPs. We found that the CHOP-ERO1α-caspase-dependent apoptotic signaling pathway was activated by upregulating the downstream target protein ERO1α and caspase-dependent mitochondrial-mediated apoptotic signaling pathway by upregulating Caspase-3 and downregulating the ratio of BCL-2/BAX. In summary, ER stress participated in cell apoptosis induced by SiNPs and CHOP regulated SiNP-induced cell apoptosis, at least partly, via activation of the CHOP-ERO1α-caspase apoptotic signaling pathway in RAW 264.7 macrophage cells.

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Cullin3 Aggravates The Inflammatory Response of PDLSCs Via Regulation of Shh Signaling and Nrf2

10.21203/rs.3.rs-368145/v1 ◽

2021 ◽

Author(s):

Wanhong Chen ◽

Jiangling Su ◽

Shixiong Cai ◽

Chun Shi

Keyword(s):

Cell Viability ◽

Cell Apoptosis ◽

Cell Counting ◽

Enzyme Linked Immunosorbent Assay ◽

Tunel Staining ◽

Western Blots ◽

Alizarin Red ◽

Shh Signaling ◽

Protein Levels ◽

Alkaline Phosphatase Staining

Abstract Objective: Sonic Hedgehog (Shh) was found to be correlated with inflammation degree of patients with periodontitis. Cullin3 is an important ubiquitin ligase for controlling Shh signaling. In this study, we exerted ourselves to clarify the roles of Shh and Cullin3 in P. gingivalis-LPS (Pg-LPS)-treated periodontal ligament stem cells (PDLSCs). Methods: Cell viability was detected using cell counting kit-8 (CCK-8). The inflammatory cytokines of PDLSCs were estimated by enzyme-linked immunosorbent assay (ELISA). The protein levels of Shh, Gli1 and NF-E2-related factor2 (Nrf2) were determined via western blots. Alkaline phosphatase staining and Alizarin red staining were performed to evaluate the differentiation and mineralization capabilities of PDLSCs. The apoptotic cells were screened by TUNEL staining. Results: Pg-LPS inhibited cell viability and triggered inflammation of PDLSCs. Overexpression of Cullin3 impeded the differentiation and mineralization capabilities of PDLSCs. Moreover, Cullin3 overexpression aggravated inflammation and cell apoptosis induced by Pg-LPS. Of note, while the protein levels of Shh, Gli1 and Nrf2 were elevated in PDLSCs treated with Pg-LPS, overexpression of Cullin3 decreased the expressions of them. Conclusion: Shh/Gli1 and Nrf2 were involved in the inflammation and cell apoptosis of PDLSCs, which was dominated by Cullin3.

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