Alexander disease causing mutations in the C-terminal domain of GFAP are deleterious both to assembly and network formation with the potential to both activate caspase 3 and decrease cell viability

Background: Histone deacetylase (HDAC) 6 inhibitors have demonstrated significant protective effects in traumatic injuries. However, their roles in neuroprotection and underlying mechanisms are poorly understood. This study sought to investigate the neuroprotective effects of Tubastatin A (Tub-A), an HDAC6 inhibitor, during oxygenglucose deprivation (OGD) in HT22 hippocampal cells. Methods: HT22 hippocampal cells were exposed to OGD. Cell viability and cytotoxicity were assessed by cell counting kit-8 (CCK-8) and lactate dehydrogenase (LDH) release assay. Cellular apoptosis was assessed by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Mitochondria membrane potential was detected using JC-1 dye. Expressions of acetylated α-tubulin, α-tubulin, cytochrome c, VDAC, Bax, Bcl- 2, cleaved caspase 3, phosphorylated Akt, Akt, phosphorylated GSK3β and GSK3β were analyzed by Western blot analysis. Results: Tub-A induced acetylation of α-tubulin, demonstrating appropriate efficacy. Tub-A significantly increased cell viability and attenuated LDH release after exposure to OGD. Furthermore, Tub-A treatment blunted the increase in TUNEL-positive cells following OGD and preserved the mitochondrial membrane potential. Tub-A also attenuated the release of cytochrome c from the mitochondria into the cytoplasm and suppressed the ratio of Bax/Bcl-2 and cleaved caspase 3. This was mediated, in part, by the increased phosphorylation of Akt and GSK3β signaling pathways. Conclusion: HDAC 6 inhibition, using Tub-A, protects against OGD-induced injury in HT22 cells by modulating Akt/GSK3β signaling and inhibiting mitochondria-mediated apoptosis.

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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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Amygdalin promotes the activity of T cells to suppress the progression of HBV-related hepatocellular carcinoma via the JAK2/STAT3 signaling pathway

BMC Infectious Diseases ◽

10.1186/s12879-020-05713-0 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Ruoyu Wang ◽

Dong Zhang ◽

Kewei Sun ◽

Jianping Peng ◽

Wenfang Zhu ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Cell Viability ◽

Caspase 3 ◽

Invasion And Migration ◽

Ifn Γ ◽

And Migration ◽

Cellular Immune

Abstract Background Hepatitis B virus (HBV) infection is a high-risk factor of hepatocellular carcinoma (HCC). Cellular immune responses are essential for HCC development, and the CD4+ and CD8+ T subtypes are identified as the primary anti-tumor immune cells. In the study, we investigated the effect and mechanism of amygdalin in the cellular immune response in HBV-related HCC and HCC progression. Methods The cell proliferation was examined by MTT analysis. Cells metastasis ability was detected by Invasion and migration assays. Quantification of apoptotic cells was performed with Flow cytometer assay. The protein levels of p-STAT3, STAT3, p-JAK2, JAK2, caspase-3, cleaved caspase-3 were detected by performing immunoblotting assays. Results We demonstrate that amygdalin treatment could rescue the HBV-T cell viability and IFN-γ and TNF-αproduction. In HBV-T cells, the MFI levels of CD8+ are lower than that in NC-T cells. Moreover, the phosphorylation levels of STAT3 and JAK2 are higher in HBV-T cells, compared to those in NC-T cells, and then reduced by amygdalin treatment. Co-culture with HBV-T cells could reduce IFN-γ and TNF-α, production while increase IL-6 and IL-10 production in HepG2.2.15 cells; these alterations could be partially reversed by amygdalin pretreatment. Finally, co-culture with HBV-T cells significantly promoted the cell viability, inhibited the apoptosis, and promoted the migration of HepG2.2.15 cells, and these alterations could be partially reversed by amygdalin treatment. Conclusion Our findings provide a rationale for further studies on the functions and mechanism of amygdalin inhibiting HBV-related HCC cell proliferation, invasion, and migration via T cell-mediated tumor immunity.

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Elevated lymphotoxin-α (TNFβ) is associated with intervertebral disc degeneration

BMC Musculoskeletal Disorders ◽

10.1186/s12891-020-03934-7 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Zhu Guo ◽

Chensheng Qiu ◽

Christina Mecca ◽

Yang Zhang ◽

Jiang Bian ◽

...

Keyword(s):

Intervertebral Disc ◽

Cell Viability ◽

Disc Degeneration ◽

Intervertebral Disc Degeneration ◽

Caspase 3 ◽

Type Ii Collagen ◽

Type Ii ◽

Optimal Conditions ◽

Cell Degeneration ◽

Lymphotoxin Α

Abstract Background Intervertebral disc degeneration (IVDD) is a primary cause of degenerative disc diseases; however, the mechanisms underlying the degeneration remain unclear. The immunoinflammatory response plays an important role in IVDD progression. The inflammatory cytokine lymphotoxin-α (LTα), formerly known as TNFβ, is associated with various pathological conditions, while its role in the pathogenesis of IVDD remains elusive. Methods Real-time quantitative polymerase chain reaction (RT-qPCR), Western blotting (WB), and enzyme-linked immunosorbent assays were used to assess the levels of LTα in human nucleus pulposus (NP) tissues between degeneration and control groups. The plasma concentrations of LTα and C-reactive protein (CRP) were compared between healthy and IVDD patients. Rat primary NP cells were cultured and identified via immunofluorescence. Methyl-thiazolyl-tetrazolium assays and flow cytometry were used to evaluate the effects of LTα on rat NP cell viability. After NP cells were treated with LTα, degeneration-related molecules (Caspase-3, Caspase-1, matrix metalloproteinase (MMP) -3, aggrecan and type II collagen) were measured via RT-qPCR and WB. Results The levels of both the mRNA and protein of LTα in human degenerated NP tissue significantly increased. Plasma LTα and CRP did not differ between healthy controls and IVDD patients. Rat primary NP cells were cultured, and the purity of primary NP cells was > 90%. Cell experiments showed inversely proportional relationships among the LTα dose, treatment time, and cell viability. The optimal conditions (dose and time) for LTα treatment to induce rat NP cell degeneration were 5 μg/ml and 48 ~ 72 h. The apoptosis rate and the levels of Caspase-3, Caspase-1, and MMP-3 significantly increased after LTα treatment, while the levels of type II collagen and aggrecan were decreased, and the protein expression levels were consistent with their mRNA expression levels. Conclusions This study demonstrated that elevated LTα is closely associated with IVDD and that LTα may induce NP cell apoptosis and reduce important extracellular matrix (ECM) proteins, which cause adverse effects on IVDD progress. Moreover, the optimal conditions for LTα treatment to induce NP cell degeneration were determined.

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Direct Effect of Septic Plasma in Human Cell Lines Viability

Blood Purification ◽

10.1159/000494597 ◽

2018 ◽

Vol 47 (1-3) ◽

pp. 270-276

Author(s):

Grazia Maria Virzì ◽

Chiara Borga ◽

Chiara Pasqualin ◽

Silvia Pastori ◽

Alessandra Brocca ◽

...

Keyword(s):

Cytochrome C ◽

Cell Viability ◽

Cell Lines ◽

Caspase 3 ◽

Test Group ◽

Organ Injury ◽

Enzyme Linked Immunosorbent Assay ◽

Caspase 9 ◽

Renal Tubular Cells

Background: Sepsis is a life-threatening condition often associated with a high incidence of multiple organs injury. Several papers suggested the immune response by itself, with the production of humoral inflammatory mediators, is crucial in determining organ injury. However, little is known of how sepsis directly induces organ injury at the cellular levels. To assess this point, we set up an in vitro study to investigate the response of renal tubular cells (RTCs), monocytes (U937) and hepatocytes (HepG2) after 24 h-incubation with septic patients’ plasma. Methods: We enrolled 26 septic patients (“test” group). We evaluated cell viability, apoptosis and necrosis by flow cytometer. Caspase-3,-8,-9 and cytochrome-c concentrations have been analyzed using the Human enzyme-linked immunosorbent assay kit. Results: We found that a decrease of cell viability in all cell lines tested was associated to the increase of apoptosis in RTCs and U937 (p < 0.0001) and increase of necrosis in HepG2 (p < 0.5). The increase of apoptosis in RTCs and U937 cells was confirmed by higher levels of caspase-3 (p < 0.0001). We showed that apoptosis in both RTCs and U937 was triggered by the activation of the intrinsic pathway, as caspase-9 and cytochrome-c levels significantly increased (p < 0.0001), while caspase-8 did not change. This assumption was strengthened by the significant correlation of caspase-9 with both cytochrome-c (r = 0.73 for RTCs and r = 0.69 for U937) and caspase-3 (r = 0.69 for RTCs and r = 0.63 for U937). Conclusion: Humoral mediators in septic patients’ plasma induce apoptosis. This fact suggests that apoptosis inhibitors should be investigated as future strategy to reduce sepsis-induced organ damages.

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Concentration-dependent effect of fumonisin B1 on apoptosis in oesophageal cancer cells

Human & Experimental Toxicology ◽

10.1177/0960327117735570 ◽

2017 ◽

Vol 37 (7) ◽

pp. 762-771 ◽

Cited By ~ 1

Author(s):

RB Khan ◽

A Phulukdaree ◽

AA Chuturgoon

Keyword(s):

Cell Viability ◽

Oesophageal Cancer ◽

Caspase 3 ◽

Quantitative Polymerase Chain Reaction ◽

Fumonisin B1 ◽

Annexin V ◽

Tail Length ◽

Bax Protein ◽

Genotoxic Carcinogens ◽

Parp 1

The geographical distribution of oesophageal cancer is linked to the exposure of fumonisin B1 (FB1), a mycotoxin produced by fungi that contaminates staple food worldwide. Non-genotoxic carcinogens like FB1 disturb homeostasis through increased cell proliferation or suppression of apoptosis. This study investigated the involvement of FB1 (0–20 μM) in spindle-shaped N-cadherin (+) CD45 (−) osteoblastic (SNO) cell death. Cell viability and death were assessed using the MTS and Annexin V-Fluos assays, respectively. Caspase activities were determined luminometrically and the comet assay assessed DNA damage. Induction of oxoguanine glycosylase 1 (OGG1) was measured using quantitative Polymerase Chain Reaction (qPCR), while cleaved poly (ADP-ribose) polymerase 1 (PARP-1) and Bax were determined by western blotting. Cell viability and PARP-1 cleavage were not affected by 1.25 μM FB1, but phosphatidylserine externalization, Bax protein expression, caspase activity, comet tail length and OGG1 transcripts were increased. The reduced cell viability in 10 μM FB1-treated cells was accompanied by corresponding increases in externalized phosphatidylserine, Bax, caspase-3/7 activity and cleaved PARP-1. The OGG1 transcripts were not significantly increased, but comet tails were increased. Bax, caspase-3/7 activities and cleaved PARP-1 were inhibited at 20 μM FB1. In addition, the OGG1 transcript levels were decreased ( p < 0.0001) along with comet lengths ( p < 0.0001). This study showed that FB1-induced apoptosis in SNO cells may be caspase-dependent or caspase-independent; the pathway used depends on the exposure concentration.

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Salmonella SipA mimics a cognate SNARE for host Syntaxin8 to promote fusion with early endosomes

The Journal of Cell Biology ◽

10.1083/jcb.201802155 ◽

2018 ◽

Vol 217 (12) ◽

pp. 4199-4214 ◽

Cited By ~ 7

Author(s):

Pawan Kishor Singh ◽

Anjali Kapoor ◽

Richa Madan Lomash ◽

Kamal Kumar ◽

Sukrut C. Kamerkar ◽

...

Keyword(s):

Actin Polymerization ◽

Enteric Fever ◽

Caspase 3 ◽

Arginine Residue ◽

Terminal Domain ◽

Trafficking Pathway ◽

Early Endosomes ◽

N Terminus ◽

Snare Motif ◽

Salmonella Survival

SipA is a major effector of Salmonella, which causes gastroenteritis and enteric fever. Caspase-3 cleaves SipA into two domains: the C-terminal domain regulates actin polymerization, whereas the function of the N terminus is unknown. We show that the cleaved SipA N terminus binds and recruits host Syntaxin8 (Syn8) to Salmonella-containing vacuoles (SCVs). The SipA N terminus contains a SNARE motif with a conserved arginine residue like mammalian R-SNAREs. SipAR204Q and SipA1–435R204Q do not bind Syn8, demonstrating that SipA mimics a cognate R-SNARE for Syn8. Consequently, Salmonella lacking SipA or that express the SipA1–435R204Q SNARE mutant are unable to recruit Syn8 to SCVs. Finally, we show that SipA mimicking an R-SNARE recruits Syn8, Syn13, and Syn7 to the SCV and promotes its fusion with early endosomes to potentially arrest its maturation. Our results reveal that SipA functionally substitutes endogenous SNAREs in order to hijack the host trafficking pathway and promote Salmonella survival.

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Qingxin Kaiqiao Fang Inhibits Aβ25-35-Induced Apoptosis in Primary Cultured Rat Hippocampal Neuronal Cells via the p38 MAPK Pathway: An Experimental Validation and Network Pharmacology Study

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2020/9058135 ◽

2020 ◽

Vol 2020 ◽

pp. 1-14

Author(s):

Tian-Qi Wang ◽

Xiao-Xiao Lai ◽

Lu-Ting Xu ◽

Yan Shen ◽

Jian-Wei Lin ◽

...

Keyword(s):

Cell Viability ◽

P38 Mapk ◽

Caspase 3 ◽

Mapk Pathway ◽

Neuronal Cells ◽

Neuronal Morphology ◽

Cell Counting ◽

Network Pharmacology ◽

Expression Levels ◽

P38 Mapk Pathway

Qingxin kaiqiao fang (QKF), a traditional Chinese medicine compound, has been applied to treat Alzheimer’s disease (AD) for many years and has exhibited remarkable effects. However, the underlying mechanism is still not explicit. The current study aims to investigate whether QKF exerts an antiapoptotic role through the p38 MAPK pathway in the course of AD. Network pharmacology analysis was applied to study the effective components, possible therapeutic targets, and AD-related pathway of QKF. Further, the AD cell model was established using amyloid-beta (Aβ)25-35 peptide and primary hippocampal neuronal cells extracted from newborn Sprague-Dawley rats. Microtubule-associated protein-2 (MAP-2) imaging was used to detect the morphology of hippocampal neurons. Western blot (WB) analysis was applied to detect the protein expression levels of p38 MAPK, p-p38 MAPK, Bcl-2, Bax, caspase-3, and cleaved caspase-3. Cell viability and apoptosis were determined using cell counting kit-8 (CCK-8) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assays, respectively. SB203580 and U46619 were used to detect changes in cell morphology, cell viability, and apoptosis upon inhibiting or activating p38 MAPK. Our present work showed that QKF protects hippocampal neuronal morphology, enhances cell viability, and reduces the number of TUNEL-positive cells. In addition, our results showed that QKF increased the expression levels of antiapoptotic proteins and decreased the expression of proapoptotic proteins. QKF at 25 mg·mL−1 best inhibited neuronal apoptosis among the three doses of QKF by suppressing p38 MAPK activity. Collectively, QKF plays an antiapoptotic role via the p38 MAPK pathway.

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Pituitary Adenylate Cyclase Activating Polypeptide Elicits Neuroprotection Against Acute Ischemic Neuronal Cell Death Associated with NMDA Receptors

Cellular Physiology and Biochemistry ◽

10.1159/000495722 ◽

2018 ◽

Vol 51 (4) ◽

pp. 1982-1995 ◽

Cited By ~ 8

Author(s):

Yuji Kaneko ◽

Julian P. Tuazon ◽

Xunming Ji ◽

Cesario V. Borlongan

Keyword(s):

Cell Death ◽

Adenylate Cyclase ◽

Protein Expression ◽

Cell Viability ◽

Caspase 3 ◽

High Mobility ◽

High Mobility Group ◽

Expression Levels ◽

Pituitary Adenylate Cyclase ◽

Nmdar Subunits

Background/Aims: The endogenous neurotrophic peptides pituitary adenylate cyclase-activating polypeptides (PACAP-27/38) protect against stroke, but the molecular mechanism remains unknown. Methods: Primary rat neural cells were exposed to PACAP-27 or PACAP-38 before induction of experimental acute ischemic stroke via oxygen-glucose deprivation-reperfusion (OGD/R) injury. To reveal PACAP’s role in neuroprotection, we employed fluorescent live/dead cell viability and caspase 3 assays, optical densitometry of mitochondrial dehydrogenase and cell growth, glutathione disulfide luciferase activity, ELISA for high mobility group box1 extracellular concentration, ATP bioluminescence, Western blot analysis of PACAP, NMDA subunits, apoptosis regulator Bcl-2, social interaction hormone oxytocin, and trophic factor BDNF, and immunocytochemical analysis of PACAP. Results: Both PACAP-27 and PACAP-38 (PACAP-27/38) increased cell viability, decreased oxidative stress-induced cell damage, maintained mitochondrial activity, prevented the release of high mobility group box1, and reduced cytochrome c/caspase 3-induced apoptosis. PACAP-27/38 increased the protein expression levels of BDNF, Bcl-2, oxytocin, and precursor PACAP. N-methyl-D-aspartate receptor (NMDAR)-induced excitotoxicity contributes to the cell death associated with stroke. PACAP-27/38 modulated the protein expression levels of NMDAR subunits. PACAP-27/38 increased the protein expression levels of the GluN1 subunit, and decreased that of the GluN2B and GluN2D subunits. PACAP-27, but not PACAP-38, increased the expression level of the GluN2C subunit. Conclusion: This study provides evidence that PACAP regulated NMDAR subunits, affording neuroprotection after OGD/R injury.

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Effect of Hydroalcoholic Extract of Ephedramajor, Memordia charantia, and Resveratrol on Cytotoxicity and Caspase-3 Genes Expression Level in MCF-7 Breast Cancer Cell Line

Gene Cell and Tissue ◽

10.5812/gct.110658 ◽

2021 ◽

Vol In Press (In Press) ◽

Author(s):

Seyed Kazem Sabbagh ◽

Ehsan Ghodrati ◽

Alireza Hajibeiki ◽

Mahta Mazaheri ◽

Mohammad Reza Sarafraz Ardakani ◽

...

Keyword(s):

Gene Expression ◽

Medicinal Plants ◽

Cell Viability ◽

Cell Line ◽

Plant Extracts ◽

Caspase 3 ◽

Cell Toxicity ◽

Hydroalcoholic Extract ◽

Expression Levels ◽

Mcf 7

Background: To increase the therapeutic effect of drugs to combat diseases, combination therapy with current chemical drugs and new medicines derived from medicinal plants is necessary. Objectives: The present work aimed to investigate the effect of hydroalcoholic extract of two medicinal plants, Ephedra major and Momordi cacharantia (Carla), and resveratrol drug on cell viability and expression levels of caspase-3 gene in MCF-7 cell line. Methods: In this experimental study, the hydroalcoholic extraction of tested plants was done with a Soxhlet extractor. The MTT assay and real-time PCR were used to determine cell toxicity and caspase-3 gene expression levels, respectively. Results: The highest and lowest cytotoxic effects of plant extracts and resveratrol were observed at concentrations of 500 and 150 µg/mL, respectively. The highest level of the caspase-3 gene expression was observed after 72 h of incubation by different concentrations of plant extracts and resveratrol. Conclusions: It can be concluded that both plant extracts could influence cell viability in MCF-7 cells via the increase of cell toxicity and expression of caspase3 gene. Thus, these species could be used in the pharmaceutical industry.

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