scholarly journals Increased expression of six-large extracellular vesicle-derived miRNAs signature for nonvalvular atrial fibrillation

2022 ◽  
Vol 20 (1) ◽  
Author(s):  
Panjaree Siwaponanan ◽  
Pontawee Kaewkumdee ◽  
Wilasinee Phromawan ◽  
Suthipol Udompunturak ◽  
Nusara Chomanee ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Flow Cytometry ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cell Activation ◽  
Target Genes ◽  
Digital Pcr ◽  
Extracellular Vesicle ◽  
Nonvalvular Atrial Fibrillation

Abstract Backgrounds Non-valvular atrial fibrillation (AF) is the most common type of cardiac arrhythmia. AF is caused by electrophysiological abnormalities and alteration of atrial tissues, which leads to the generation of abnormal electrical impulses. Extracellular vesicles (EVs) are membrane-bound vesicles released by all cell types. Large EVs (lEVs) are secreted by the outward budding of the plasma membrane during cell activation or cell stress. lEVs are thought to act as vehicles for miRNAs to modulate cardiovascular function, and to be involved in the pathophysiology of cardiovascular diseases (CVDs), including AF. This study identified lEV-miRNAs that were differentially expressed between AF patients and non-AF controls. Methods lEVs were isolated by differential centrifugation and characterized by Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM), flow cytometry and Western blot analysis. For the discovery phase, 12 AF patients and 12 non-AF controls were enrolled to determine lEV-miRNA profile using quantitative reverse transcription polymerase chain reaction array. The candidate miRNAs were confirmed their expression in a validation cohort using droplet digital PCR (30 AF, 30 controls). Bioinformatics analysis was used to predict their target genes and functional pathways. Results TEM, NTA and flow cytometry demonstrated that lEVs presented as cup shape vesicles with a size ranging from 100 to 1000 nm. AF patients had significantly higher levels of lEVs at the size of 101–200 nm than non-AF controls. Western blot analysis was used to confirm EV markers and showed the high level of cardiomyocyte expression (Caveolin-3) in lEVs from AF patients. Nineteen miRNAs were significantly higher (> twofold, p < 0.05) in AF patients compared to non-AF controls. Six highly expressed miRNAs (miR-106b-3p, miR-590-5p, miR-339-3p, miR-378-3p, miR-328-3p, and miR-532-3p) were selected to confirm their expression. Logistic regression analysis showed that increases in the levels of these 6 highly expressed miRNAs associated with AF. The possible functional roles of these lEV-miRNAs may involve in arrhythmogenesis, cell apoptosis, cell proliferation, oxygen hemostasis, and structural remodeling in AF. Conclusion Increased expression of six lEV-miRNAs reflects the pathophysiology of AF that may provide fundamental knowledge to develop the novel biomarkers for diagnosis or monitoring the patients with the high risk of AF.

Nutlin-3A, an MDM2 Antagonist, Induces p53-Dependent Cell Cycle Arrest and Apoptosis in Hodgkin Lymphoma (HL).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2506-2506
Author(s):  
Elias Drakos ◽  
Athanasios Thomaides ◽  
Jiang Li ◽  
Marina Konopleva ◽  
L. Jeffrey Medeiros ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Death ◽  
Western Blot ◽  
Cell Cycle Arrest ◽  
Small Molecule ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
P53 Gene ◽  
Cycle Arrest

Abstract p53 is the most frequently mutated tumor suppressor gene in human cancer. However, in Hodgkin lymphoma (HL) p53 is mutated only in a small subset of cases suggesting that modulation of wild-type-p53 (wt-p53) levels in Hodgkin and Reed-Sternberg (HRS) cells may have therapeutic implications in these patients. MDM2 (HDM2 in humans) is a physiologic negative regulator of p53 levels through a well-established auto-regulatory feedback loop. Nutlin-3A is a recently developed small molecule, which antagonizes mdm2 through disruption of p53-MDM2 interaction resulting in p53 stabilization. We hypothesized that nutlin 3A may stabilize p53 in HRS cells carrying wt-p53 gene, thus leading to p53-dependent apoptosis and G1-S cell cycle arrest. We used two novel classical HL cell lines recently established in our Institution, MDA-V and MDA-E, which have been shown to carry wt-p53 gene. As a control, we used a HL cell line L-428 harboring a mutant p53 (mt-p53) gene product (deletion at exon 4). We investigated effects on apoptosis and cell cycle arrest after treatment of cultured HRS cells with nutlin-3A or a 150-fold less active enantiomere, nutlin-3B. Treatment with nutlin-3A resulted in substantial cell death (up to 65%) in a concentration-dependent manner associated with increased apoptosis as shown by apoptotic morphology (DAPI immunofluorescence), annexin V binding (flow cytometry) and caspase activation (Western blot analysis) in MDA-V and MDA-E cells, but not in L-428 cells. Nutlin-3A-induced apoptotic cell death was accompanied by stabilization of p53 protein as detected by western blot analysis and immunofluorescence and up-regulation of pro-apoptotic Bax, a known target of p53. Inhibition of nuclear export by leptomycin B stabilized p53 at a similar level as compared to nutlin-3A treatment in these cells, suggesting that nutlin-3A stabilized p53 through inhibition of MDM2-mediated degradation of the protein. By contrast, no changes in cell viability, growth or apoptosis were seen after treatment with the inactive nutlin-3B small molecule. Treatment with nutlin-3A also resulted in a significant decrease (up to 85%) of cells in S-phase and a dose-dependent increase of cells in G1 phase of cell cycle as detected by flow cytometry, in MDA-V and MDA-E cells, but not in L-428 cells. Cell cycle arrest was associated with up-regulation of the cyclin-dependent kinase inhibitor p21, a transcriptional target of p53. In contrast, treatment of HRS cells with nutlin-3B had no effects on the cell cycle irrespective of p53 mutation status. Furthermore, combined treatment with nutlin-3A and doxorubicin revealed synergistic effects and enhanced cytotoxicity in HRS cells with wt-p53 gene. Targeting MDM2 with the specific antagonist nutlin-3A that leads to non-genotoxic p53 activation, apoptosis induction and cell cycle inhibition may provide a new therapeutic approach for patients with HL.

Suppression of Interferon Regulatory Factor 8 Modulates Toll Like Receptor 2 and Toll Like Receptor 7/8 Induced Dendritic Cell Activation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2573-2573
Author(s):  
Daniela Werth ◽  
Anita Bringmann ◽  
Katharina Brauer ◽  
Karin von Schwarzenberg ◽  
Stefanie Held ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cell Activation ◽  
Nuclear Translocation ◽  
Interferon Regulatory Factor ◽  
Toll Like Receptor ◽  
Regulatory Factor ◽  
Dendritic Cell Activation

Abstract Interferon regulatory factor 8 (IRF-8) is a member of the IRF family of transcription factors, which are stimulated through interferon mediated pathways. In mice, IRF-8 seems to play an essential role in the development and maturation of dendritic cells (DCs). However, very limited knowledge is available about the potential role of IRF-8 in the human system. To bridge this gap we analyzed function and activation of human monocyte-derived dendritic cells (mDCs) lacking IRF-8 expression. To knockdown IRF-8 protein levels, we electroporated mDCs with different siRNAs against IRF-8. Additionally, we stimulated the electroporated mDCs with the Toll like receptor (TLR) 2 ligand Pam3Cys or the TLR 7/8 ligand R848. IRF-8 knockdown in mDCs was verified constantly by Western Blot analysis using an anti-IRF-8 antibody. We found that IRF-8 knockdown clearly diminished the expression of the human lymphocyte antigen molecules HLA-ABC and HLA-DR in Pam3Cys and R848 stimulated mDCs. To gain functional data, we performed ELISAs to determine cytokine and chemokine secretion. The electroporation of mDCs with IRF-8 specific siRNA resulted in profound inhibition of secretion of the cytokines IL-6, IL-12 and TNF-a as well as the chemokines MIP-1a (CCL3), MCP-1 (CCL2) and RANTES (CCL5). To get additional information on IRF-8 function in human mDCs, the regulation of signal transduction pathways was determined by Western Blot analysis. The suppression of IRF-8 diminished the nuclear translocation of the NF-kB family member’s c-Rel and RelB as well as PU.1 and IRF-3 in activated mDCs. In addition, we showed that the suppression of IRF-8 caused a reduced phosphorylation of ERK and JNK, but had no effect on the expression of STAT3. In summary, the knockdown of IRF-8 reduced the capability of mDCs to develop appropriate phenotype and functions in response to activating stimuli. Our results indicate that these effects are mediated via the ERK, NF-kB and PU.1 signalling pathways. IRF-8 plays an important role in the activation and function of human mDCs.

Monoclonal Antibody Against ROR1 in Chronic Lymphocytic Leukemia Cells Induced Apoptosis Via PI3-Kinase/AKT/CREB Pathway

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1769-1769
Author(s):  
Amir Hossein Daneshmanesh ◽  
Mohammad Hojjat-Farsangi ◽  
Asa Sandin ◽  
Abdul Salam Khan ◽  
Ali Moshfegh ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Signaling Pathways ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Pi3 Kinase ◽  
Signaling Proteins ◽  
Downstream Signaling ◽  
Induced Apoptosis ◽  
Creb Pathway

Abstract Abstract 1769 Background: Phosphoinositide 3-kinase (PI3K)/AKT cascade regulates cell survival, proliferation and differentiation in a variety of cells. In CLL cells PI3K pathway is constitutively activated leading to AKT activation and phosphorylation of cAMP response element-binding protein (CREB). CREB is a transcription factor overexpressed and constitutively phosphorylated in a variety of cancers and seems to have a role in tumor pathobiology. There is a great need to develop novel strategies for targeted therapy in CLL. Monoclonal antibodies (mAbs) specifically targeting leukemic cells might be a rewarding approach. ROR1 is a type I transmembrane receptor tyrosine kinase belonging to one of the twenty families of receptor tyrosine kinases (RTKs). ROR1 is overexpressed on CLL cells but not in white blood cells of healthy donors. ROR1 is constitutively phosphorylated in CLL and siRNA transfection induced apoptosis. We have developed a unique anti-ROR1 mAb directed against CRD (cysteine-rich domain) of the extracellular region of ROR1 capable of inducing direct apoptosis of primary CLL cells. Our anti-CRD mAb induced dephosphorylation of the ROR1 molecule. Aims: To study the apoptotic effect of an anti-ROR1 CRD mAb and effects on downstream signaling pathways involved in CLL, specially the PI3-kinase/AKT/CREB pathway using primary CLL cells. Methods: Using a peptide-based mouse mAb generation method we produced several mAbs against the three extracellular domains of ROR1. In the current study we used one of the best anti-ROR1 antibodies, an anti-CRD mAb raised against the CRD region of ROR1 (Daneshmanesh et al., Leukemia. 2012 Jun;26(6):1348-55). Flow cytometry was used for surface staining of ROR1. Primary CLL cells were incubated with the anti-ROR1 CRD mAb and apoptosis was detected by the MTT assay and Annexin V/propidium iodide (flow cytometry) methods in a 24 h assay. Antibody untreated and treated cell lysates were prepared and subjected to Western blot analysis for identification of signaling molecules involved in apoptosis induced by the anti-ROR1 CRD mAb. We analysed total and phosphorylated levels of the following signaling proteins: AKT, p-AKT, PI3K, p-PI3K, CREB, p-CREB, ERK, p-ERK, PKC and p-PKC. Phosphoproteins were measured before incubation with the mAb and after 20 min-2 h. Results: ROR1 surface expression was detected on 80–85% of the CLL cells. The frequency of apoptotic cells induced by the anti-CRD mAb was in the range of 45–50% which is in accordance with our previous reports (see above). Time kinetics experiments using anti-ROR1 CRD mAb incubated with primary CLL cells revealed dephosphorylation of ROR1 downstream signaling molecules. We analysed the following molecules known to be involved in CLL: PKC, PI3-kinase and ERK1/2. After co-culturing CLL cells with the anti-ROR1 CRD mAb, Western blot analysis showed decreased level of phosphorylated AKT in treated compared to untreated samples. No changes in the phosphorylation levels of ERK1/2 and PKC proteins were seen. Furthermore, we analysed the PI3-kinase protein which is upstream of AKT, and noticed that in CLL cells treated with the anti-ROR1 CRD mAb, the phosphorylation intensity of PI3-kinase p85 isoform has decreased but not p55 isoforrn. Moreover, we also studied the CREB phosphorylation in treated and untreated CLL samples and detected dephosphorylation of CREB in treated as compared to untreated samples. Conclusion: Incubation of CLL cells with an anti-ROR1 CRD mAb induced apoptosis of primary CLL cells. Apoptosis was preceded by dephosphorylation within 2 h of PI3-kinase, AKT and CREB proteins indicating deactivation of these signaling proteins by the anti-ROR1 mab. In untreated CLL cells no effect on phosphorylation of these proteins was noted. Furthermore our ROR1 mAb did not dephosphorylate PKC or ERK. Our data may suggest that activation of CREB molecule might occur via the PI3K/AKT pathway and may be a survival signal in CLL cells associated with the aberrant expression of ROR1. The constitutive phosphorylation of PKC and ERK1/2 seen in CLL might not be related to the overexpression of ROR1. Further studies are warranted for a better understanding of signaling pathways associated with ROR1 and the downstream signaling effects of ROR1 targeting drugs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Acetylshikonin Suppresses Diffuse Large B-Cell Lymphoma Cell Growth By Targeting The TOPK Signaling Pathway

2021 ◽  
Author(s):  
Jieke Cui ◽  
Rong Guo ◽  
Yingjun Wang ◽  
Yue Song ◽  
Xuewen Song ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Growth ◽  
Western Blot ◽  
B Cell ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Background: Diffuse large B-cell lymphoma (DLBCL) is one of the most common causes of cancer death worldwide, and responds badly to the existing treatment. Thus, identifying the novel therapeutic targets of DLBCL are urgent. Methods and results: In this study, we found that the T-lymphokine-activated killer cell-originated protein kinase (TOPK) was highly expressed in DLBCL cells and tissues. The TOPK expression were analyzed by bioinformatics analysis, immunohistochemistry (IHC) and western blot analysis. TOPK knockdown inhibited cell growth and induced apoptosis of DLBCL cells with MTS and flow cytometry. Further experiments demonstrated that acetylshikonin, the targeted compound of TOPK, could attenuate the cell growth and aggravate the cell apoptosis through TOPK/extra cellular signal-regulated kinase (ERK)-1/2 signaling using MTS, flow cytometry and western blot analysis. In addition, we demonstrated that TOPK overexpression significantly reduced the acetylshikonin effect on cell proliferation and apoptosis in U2932 and OCI-LY8 cells using MTS, flow cytometry and western blot analysis. Conclusions: Taken together, the present study suggests that the targeted inhibition of TOPK by acetylshikonin may be a promising approach to the treatment of DLBCL.

scholarly journals Ovarian Cancer HO-8910 Cell Apoptosis Induced by Crocin in vitro

2015 ◽  
Vol 10 (2) ◽  
pp. 1934578X1501000 ◽  
Author(s):  
Dan Xia
Keyword(s):  
Ovarian Cancer ◽  
Flow Cytometry ◽  
Western Blot ◽  
Blot Analysis ◽  
Cell Apoptosis ◽  
Western Blot Analysis ◽  
Caspase 3 ◽  
Cell Cycle Distribution ◽  
Apoptotic Pathway ◽  
Apoptosis Rate

The effect and mechanism of ovarian cancer HO-8910 cell apoptosis induced by crocin. MTT assay was performed to detect the inhibitory action of crocin on the proliferation of HO-8910 cells. Flow cytometry was used to test the cell cycle distribution and apoptosis rate of ovarian cancer HO-8910 cells. Western blot analysis was utilized to measure the levels of apoptotic proteins such as p53, Fas/APO-1, and Caspase-3. MTT analysis revealed that crocin significantly inhibited the growth of HO-8910 cells. Additionally, flow cytometry illustrated that crocin raised the proportion of HO-8910 cells in the G0/G1 phase and increased their apoptosis rate. Furthermore, Western blot analysis revealed that crocin up-regulated the expression of p53, Fas/APO-1, and Caspase-3. The results of this study showed that crocin can significantly inhibit the growth of HO-8910 cells and arrest them in the G0/G1 phase. Crocin can also promote ovarian cancer HO-8910 cell apoptosis, most likely by increasing p53 and Fas/APO-1 expression, and then activating the apoptotic pathway regulated by Caspase-3.

Trafficking of apical proteins into clathrin-coated vesicles isolated from rat renal cortex

1994 ◽  
Vol 266 (4) ◽  
pp. F554-F562 ◽  
Author(s):  
T. G. Hammond ◽  
P. J. Verroust
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Renal Cortex ◽  
Coated Vesicles ◽  
Brush Border Enzymes ◽  
Molecular Weights ◽  
Endosomal Pathway ◽  
Fluorescent Dextran

The endosomal pathway of the rat renal cortex was labeled by intravenous infusion of fluorescent dextran small enough to cross the glomerular ultrafiltration barrier and be taken up by luminal endocytosis in the proximal tubule. Clathrin-coated vesicles (CCV) were isolated from the rat renal cortex utilizing discontinuous sucrose density gradients and negative lectin selection. More than 99 +/- 1% (n = 4) of the isolated vesicles contain entrapped fluorescein dextran when analyzed by small-particle flow cytometry techniques. Similarly, flow cytometry analysis demonstrates brisk H(+)-adenosinetriphosphatase activity in virtually all the vesicles. Western blot analysis of the vesicle proteins with a polyclonal anticlathrin antibody stains bands consistent with clathrin and adaptins. When the isolated vesicles are decoated by exposure to 0.5 M tris(hydroxymethyl)aminomethane, the proteins released match the molecular weights of the proteins identified on Western blot analysis. Flow cytometry demonstration of brush border enzymes in > 99% of the vesicles and Western blot identification of maltase suggests both that these vesicles are of apical origin and that apical enzymes traffic into endosomal elements. Additionally, two glycoproteins detectable in this fraction on Western blot analysis and flow cytometry immunocytochemistry are derived from intermicrovillar clefts traffic into the endosomal pathway. Hence, apical proteins traffic into a population of CCV isolated from the rat renal cortex.

Neferine-induced apoptosis is dependent on the suppression of Bcl-2 expression via downregulation of p65 in renal cancer cells

2019 ◽  
Vol 51 (7) ◽  
pp. 734-742 ◽  
Author(s):  
Eun-Ae Kim ◽  
Eon-Gi Sung ◽  
In-Hwan Song ◽  
Joo-Young Kim ◽  
Hwa-Jung Sung ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Renal Cancer ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cancer Cell Lines ◽  
Induced Apoptosis

Abstract Neferine is an alkaloid extracted from a seed embryo of Nelumbo nucifera and has recently been shown to have anticancer effects in various human cancer cell lines. However, the detailed molecular mechanism of neferine-induced apoptosis has not been elucidated in renal cancer cells. In the present study, we observed that neferine induced inhibition of cell proliferation and apoptosis in Caki-1 cells in a dose-dependent manner by using MT assay and flow cytometry and that neferine-mediated apoptosis was attenuated by pretreatment with N-benzyloxycarbony-Val-Ala-Asp (O-methyl)-fluoromethyketone, a pan-caspase inhibitor. Treatments with neferine dose-dependently downregulated B cell lymphoma-2 (Bcl-2) expression at the transcriptional level determined by reverse transcriptase-polymerase chain reaction. The forced expression of Bcl-2 and p65 attenuated the neferine-mediated apoptosis in Caki-1 cells. In addition, neferine induced apoptosis by downregulating Bcl-2 and p65 expression in the other two kidney cancer cell lines determined by flow cytometry and western blot analysis. Finally, we observed that treatment with neferine induced apoptosis by inhibiting the NF-κB pathway through caspase-mediated cleavage of the p65 protein by western blot analysis. Collectively, this study demonstrated that neferine-induced apoptosis is mediated by the downregulation of Bcl-2 expression via repression of the NF-κB pathway in renal cancer cells.

scholarly journals ADSC Exosomes Mediate lncRNA-MIAT Alleviation of Endometrial Fibrosis by Regulating miR-150-5p

2021 ◽  
Vol 12 ◽  
Author(s):  
Xiaowen Shao ◽  
Jinlong Qin ◽  
Chendong Wan ◽  
Jiajing Cheng ◽  
Lian Wang ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Target Genes ◽  
Expression Level ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Injury Model ◽  
Gene Assay ◽  
Tgf Β1

BackgroundSecondary infertility remains a major complication of endometrial fibrosis in women. The use of exosomes from adipose-derived mesenchymal stem cells (ADSCs) has shown promising results for the treatment of endometrial fibrosis. However, the mechanisms of action of ADSC-exosome (ADSC-Exo) therapy remain unclear.Materials and MethodsAn endometrial fibrosis model was established in mice treated with alcohol and endometrial epithelial cells (ESCs) treated with TGF-β1. ADSCs were isolated from Sprague Dawley (SD) rats, and exosomes were isolated from ADSCs using ExoQuick reagent. Exosomes were identified by transmission electron microscopy (TEM), NanoSight, and Western blot analysis. The expression level of lncRNA-MIAT was detected by qPCR analysis. Western blot analysis was carried out to determine the protein levels of fibrosis markers (TGFβR1, α-SMA, and CK19). A dual-luciferase reporter gene assay was used to verify the relationship between target genes. The endometrial tissues of the endometrial fibrosis model were stained with HE and Masson’s trichrome.ResultsADSCs and ADSC-Exos were successfully isolated, and the expression level of lncRNA-MIAT was significantly down-regulated in endometrial tissue and the TGF-β1-induced ESC injury model, whereas ADSC-Exos increased the expression of lncRNA-MIAT in the TGF-β1-induced ESC model. Functionally, ADSC-Exo treatment repressed endometrial fibrosis in vivo and in vitro by decreasing the expression of hepatic fibrosis markers (α-SMA and TGFβR1) and increasing the expression of CK19. Moreover, miR-150-5p expression was repressed by lncRNA-MIAT in the TGF-β1-induced ESC injury model. The miR-150-5p mimic promoted TGF-β1-induced ESC fibrosis.ConclusionADSC-Exos mediate lncRNA-MIAT alleviation of endometrial fibrosis by regulating miR-150-5p, which suggests that lncRNA-MIAT from ADSC-Exos may be a viable treatment for endometrial fibrosis.

scholarly journals Chrysophanol Prevents Lipopolysaccharide-Induced Hepatic Stellate Cell Activation by Upregulating Apoptosis, Oxidative Stress, and the Unfolded Protein Response

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Jiunn-Sheng Wu ◽  
Valeria Chiu ◽  
Chou-Chin Lan ◽  
Ming-Chieh Wang ◽  
I.-Shiang Tzeng ◽  
...  
Keyword(s):  
Western Blot ◽  
Dna Fragmentation ◽  
Hepatic Stellate Cell ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cell Activation ◽  
Caspase 3 ◽  
Stellate Cell ◽  
Unfolded Protein ◽  
Protein Response

Hepatic stellate cell (HSC) activation is a vital driver of liver fibrosis. Recent research efforts have emphasized the clearance of activated HSCs by apoptosis, senescence, or reversion to the quiescent state. LPS induces human HSC activation directly and contributes to liver disease progression. Chrysophanol is an anthraquinone with hepatoprotective and anti-inflammatory effects. This study aimed to investigate the pharmacological effects and mechanisms of chrysophanol in an LPS-induced activated rat HSC cell line (HSC-T6). The fibrosis phenotype was identified from the expression of α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), and integrin β1 by western blot analysis. We examined DNA fragmentation by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. We detected the apoptotic markers p53 and cleaved caspase-3 by western blot analysis. Intracellular ROS were labeled with 2′,7′-dichlorofluorescein diacetate (DCF-DA) and the levels were measured by flow cytometry. Finally, we evaluated the ER stress markers binding immunoglobulin protein (BiP) and C/EBP homologous protein (CHOP) by Western blot analysis. Our results showed that chrysophanol decreased HSC-T6 cell viability in LPS-induced activated HSCs. Chrysophanol increased the expression of α-SMA, CTGF, integrin βI, p53, cleaved caspase-3, and DNA fragmentation. Chrysophanol also elevated ROS levels and increased the expression of BiP and CHOP. Pretreatment with chrysophanol prevented LPS-induced HSC-T6 cell activation by upregulating apoptosis, ROS accumulation, unfolded protein response (UPR) activation, and the UPR proapoptotic effect.

Rhamnazin Ameliorates Traumatic Brain Injury in Mice via Reduction in Apoptosis, Oxidative Stress, and Inflammation

2021 ◽  
pp. 1-8
Author(s):  
Boxiao Yang ◽  
Rui Zhang ◽  
Qire Sa ◽  
Yanli Du
Keyword(s):  
Oxidative Stress ◽  
Traumatic Brain Injury ◽  
Flow Cytometry ◽  
Brain Injury ◽  
Western Blot ◽  
Protective Effect ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Neuronal Degeneration ◽  
Flow Cytometry Analysis

<b><i>Background:</i></b> Traumatic brain injury (TBI) is posing serious health challenges for people across the globe due to high morbidity and mortality. However, none of the agents prevents or limits the damage caused by TBI because of its multifactorial etiology. Thus, the discovery of novel agents which can act via several pathways could serve the purpose and afford favorable consequence against TBI. Therefore, in the present article, we intended to investigate the protective effect of rhamnazin (RMZ), a dimethoxyflavone against experimentally induced TBI in mice. <b><i>Methods:</i></b> The effect of RMZ was investigated on cerebral edema and grip test score after induction of experimental brain injury in rats. The effect of RMZ was also investigated on neuronal degeneration in brain tissues of the experimental mice via Nissl staining and flow cytometry analysis. The expression of Bax and Bcl-2 was also quantified using Western blot analysis. The level of inflammatory cytokines (TNF-α and IL-1β) and oxidative stress markers (malondialdehyde, superoxide dismutase, and glutathione peroxidase) was also determined using enzyme-linked immunosorbent assay. <b><i>Results:</i></b> RMZ showed a significant reduction in edema and improved grip strength. It also prevented neuronal degeneration via inhibition of neuronal apoptosis as shown by flow cytometry analysis. RMZ showed an antiapoptotic effect via reduction of Bax and increased the expression of Bcl-2 in Western blot analysis. It also showed to inhibit oxidative stress and inflammation compared to the TBI group. <b><i>Conclusion:</i></b> Collectively, our study is first to demonstrate the protective effect of RMZ against experimentally induced TBI in rats.

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