scholarly journals Comparison of CRISPR and adenovirus-mediated Myd88 knockdown in RAW 264.7 cells and responses to lipopolysaccharide stimulation

2021 ◽  
Vol 8 (3) ◽  
pp. e151
Author(s):  
Alexander L. Kolb ◽  
Marinaliz Reynoso ◽  
Ronald W. Matheny
Keyword(s):  
Genetic Manipulation ◽  
Interleukin 10 ◽  
Primary Response ◽  
Raw 264.7 Cells ◽  
Raw 264.7 ◽  
Necrosis Factor Alpha ◽  
Raw 264.7 Macrophages ◽  
Inflammatory Stimuli ◽  
Adenoviral Transduction ◽  
Nfκb Pathway

Genomic manipulation offers the possibility for novel therapies in lieu of medical interventions in use today. The ability togenetically restore missing inflammatory genes will have a monumental impact on our current immunotherapy treatments. This study compared the efficacy of two different genetic manipulation techniques: clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) transfection to adenoviral transduction to determine which method would provide the most transient and stable knockdown of myeloid differentiation primary response 88 (MyD88). MyD88 is a major regulator of nuclear factor kappa light chain enhancer of activated B cells (NFκB) pathway in Raw 264.7 macrophages. Following genetic manipulation, cells were treated for 24 h with Lipopolysaccharide (LPS) to stimulate the inflammatory pathway. Confirmation of knockdown was determined by western immunoblotting and quantification of band density. Both CRISPR/Cas9 and adenoviral transduction produced similar knockdown efficiency (~64% and 60%, respectively) in MyD88 protein 48 h post adenoviral transduction. NFκB phosphorylation was increased in CRISPR/Cas9-mediated MyD88 knockdown and control cells, but not in adenovirus-mediated MyD88 knockdown cells, following LPS administration. CRISPR/Cas9-mediated MyD88 knockdown macrophages treated with LPS for 24 h showed a 65% reduction in tumor necrosis factor alpha (TNFα) secretion, and a 67% reduction in interleukin-10 (IL-10) secretion when compared to LPS-stimulated control cells (P ≤ 0.01 for both). LPS did not stimulate TNFα or IL-10 secretion in adenovirus-mediated control or MyD88 knockdown cells. These data demonstrate that Raw 264.7 macrophages maintain responsiveness to inflammatory stimuli following CRISPR/Cas9-mediated reductions in MyD88, but not following adenovirus-mediated MyD88 knockdown.

scholarly journals Tocotrienol-rich Fraction Modulated Genes Responsible for Inflammation in Lipopolysaccharide-stimulated RAW 264.7 Macrophages

2021 ◽  
pp. 106-118
Author(s):  
Sitti Rahma Abd Hafid ◽  
Maliya Azilah Mohammad Aini ◽  
Nabiha Iran ◽  
Irmaliayana Norisam ◽  
Khairul Adzfa Radzun ◽  
...  
Keyword(s):  
Fold Change ◽  
Alpha Tocopherol ◽  
In Vivo Studies ◽  
Raw 264.7 Cells ◽  
Raw 264.7 ◽  
Necrosis Factor Alpha ◽  
Raw 264.7 Macrophages ◽  
Raw264.7 Macrophages ◽  
Tocotrienol Rich Fraction

Background: Inflammation plays a vital role in the pathogenesis of chronic non-communicable diseases (NCDs), the leading health issue worldwide. An earlier study reported that tocotrienol-rich fraction (TRF) showed better anti-inflammation effects in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Aim: This study aimed to investigate the anti-inflammatory effects of tocotrienol-rich fraction at the molecular level by looking at the genes that were differentially regulated and pathways affected in LPS-stimulated macrophages exposed to TRF using the microarray approach. Methods: A microarray study was carried out in LPS-stimulated RAW 264.7 macrophages. Total ribonucleic acid (RNA) was extracted from the RAW 264.7 cells treated with TRF (10µg/mL), alpha-tocopherol (10 µg/mL) or LPS (10 ng/mL). Untreated cells served as control. Enrichment analyses, such as Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG), were conducted for genes listed in the differentially expressed genes (DEGs).  Results: The microarray analysis showed that the expression of five genes [Hamp, Interleukin-1a (IL-1a), IL-b, C-X-C motif chemokine ligand 2 (CXCL2) and colony-stimulating factor 3 (CSF3)] and one gene (SLC1A4), an amino acid transporter, was modulated (fold change 2, P< 0.05) in the TRF-treated cells. With a more stringent analysis (fold change 3, P < 0.05), only one gene (CSF3) was downregulated in the TRF-treated in RAW 264.7 cells. Analysis using the GO and KEGG pathways revealed interactions between pro-inflammatory agents such as tumor necrosis factor-alpha (TNFα) and nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-B), as well as signaling pathways of interleukin (IL)-1 and IL-17. Conclusion: TRF modulated the expression of genes responsible for acute and chronic inflammation that were part of the lipoxygenase (LOX) and cyclooxygenase (COX) inflammatory pathways. Further investigation on the effects of TRF in different cell lines and in vivo studies should be conducted in the future.

scholarly journals Suppression of inflammation by ethanol extract of Clausena lansium via modulation of TLR4/MYD88/TRAF6 signaling pathway in RAW 264.7 macrophages

2019 ◽  
Vol 17 ◽  
pp. 205873921984197
Author(s):  
Guihong Huang ◽  
Juan Li ◽  
Wanlian Li ◽  
Tianxu Liu ◽  
Guojun Jiang ◽  
...  
Keyword(s):  
Ethanol Extract ◽  
Raw 264.7 Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Raw 264.7 ◽  
Rt Pcr ◽  
Anticancer Activities ◽  
Necrosis Factor Alpha ◽  
Raw 264.7 Macrophages ◽  
Protein Levels ◽  
Tnf Α

The fruit and root of Clausena lansium are remedy for bronchitis in oriental traditional medicine. Extracts from Clausena lansium are reported to have antioxidant, anti-inflammatory, and anticancer activities. This study is designed to investigate whether the ethanol extract of Clausena lansium (Lour.) Skeels could inhibit the lipopolysaccharides (LPS)-induced inflammatory in RAW 264.7 macrophages and the underlining mechanisms. Enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR) are used to detect the secretion of tumor necrosis factor alpha (TNF-α) protein and expression of TNF-α mRNA, respectively. RT-PCR and Western blotting are used to determine the mRNA and protein levels of TLR4, MYD88, and TRAF6, respectively. The extract of Clausena lansium suppressed the expression and secretion of TNF-α, which is released by RAW 264.7 cells after LPS induction. Meanwhile, the expression of TLR4, MYD88, and TRAF6 are decreased by extracts from Clausena lansium. Meanwhile, NBP2-29328, an inhibitor of MYD88, synergistically enhances the inhibiting effect of extract from Clausena lansium. The results imply that ethanol extract from Clausena lansium attenuate macrophage-mediated inflammation and TLR4/MYD88/TRAF6 pathway is its potential target.

scholarly journals Nutritional Value and Anti-inflammation Activity of Misutkaru with Added Gryllus bimaculatus Powder

2021 ◽  
Vol 19 (3) ◽  
pp. 467-476
Author(s):  
Jung-Soon Han
Keyword(s):  
Amino Acid ◽  
Inflammatory Cytokines ◽  
Nutritional Value ◽  
Interleukin 1 ◽  
Raw 264.7 Cells ◽  
Raw 264.7 ◽  
Gryllus Bimaculatus ◽  
Necrosis Factor Alpha ◽  
Automatic Amino Acid Analyzer ◽  
Tnf Α

Purpose: This study investigated the nutritional value of Misutkaru with added Gryllus bimaculatus powder (GBM) and its applicability as a healthy functional food.Methods: Chemical analysis of the moisture, crude fat, protein, and mineral contents was performed in accordance with the Association of Official Analytical Chemists (AOAC) guidelines. The amino acid and fatty acid compositions were analyzed using an automatic amino acid analyzer and gas chromatography, respectively. The levels of inflammatory cytokines tumor necrosis factor‑alpha (TNF-α), interleukin 1 beta (IL-1β), and interleukin 6 (IL‑6) induced by lipopolysaccharides in RAW 264.7 cells were measured.Results: The general composition per 100 g of GBM was 41.87 g protein, 19.75 g fat, and 28.52 g carbohydrates. The mineral content per 100 g of GBM was 889.66 mg calcium, 1189.73 mg potassium, 220.36 mg magnesium, 207.51 mg sodium, 694.81 mg phosphorus, and 15.50 mg zinc. In particular, valine (21.361 mg/kg), leucine (29.180 mg/kg), and isoleucine (15.562 mg/kg) were abundant in GBM. GBM also effectively downregulated the production of the inflammatory cytokines TNF-α, IL-1β, and IL-6 in RAW 264.7 macrophages.Conclusion: Misutkaru with added Gryllus bimaculatus powder may have potential for application in the development of food materials or foods to prevent muscle loss in elderly individuals and sarcopenia patients, build muscle, and prevent increase in blood lipid concentrations in middle aged people. In particular, as Gryllus bimaculatus is low in fat and carbohydrates, it can be used as a diet material.

Anti-inflammatory Effects of Achillea millefolium on Atopic Dermatitis-Like Skin Lesions in NC/Nga Mice

2020 ◽  
Vol 48 (05) ◽  
pp. 1121-1140
Author(s):  
Hien T.T. Ngo ◽  
Eunson Hwang ◽  
Hyungoo Kang ◽  
Bom Park ◽  
Seul A. Seo ◽  
...  
Keyword(s):  
Atopic Dermatitis ◽  
Elevated Serum ◽  
Mitogen Activated Protein Kinase ◽  
Raw 264.7 Cells ◽  
Achillea Millefolium ◽  
Raw 264.7 ◽  
Hacat Cells ◽  
Necrosis Factor Alpha ◽  
Cox 2 ◽  
Anti Inflammatory

Achillea millefolium L. (AM) is an aromatic herb with a variety of pharmacological properties, such as anti-inflammatory and anti-allergic activities. However, AM’s effects on atopic dermatitis (AD) have not been investigated. This study evaluates the anti-AD activity of 50% ethanol-extracted AM in murine macrophage Raw 264.7 cells, in tumor necrosis factor-alpha/interferon-gamma (TNF-[Formula: see text]/IFN-[Formula: see text])-stimulated human immortal keratinocyte HaCaT cells in vitro, and in Biostir-AD-treated NC/Nga mice in vivo. The results showed that AM significantly downregulated expression of pro-inflammatory cytokines, such as INOS, COX-2, and interleukin (IL)-6 in lipopolysaccharide (LPS)-treated Raw 264.7 cells. The mRNA expressions of INOS, COX-2, and IL-6 decreased by 76.1%, 69.3%, and 31.8%, respectively. Overexpression of chemokines, such as activation-regulated chemokine and macrophage-derived chemokine, regulated on activation of normal T-cell expressed and secreted, and IL-8 was inhibited by 70.01%, 52.91%, 73.53%, and 18.93%, respectively, in TNF-[Formula: see text]/IFN-[Formula: see text]-stimulated HaCaT cells by downregulating the mitogen-activated protein kinase, I[Formula: see text]B[Formula: see text], and the signal transducer and activator of transcription 1 signaling pathways. AD-like symptoms, such as elevated serum immunoglobin E levels, epidermal thickening, high dermatitis severity score, transepidermal water loss, and reduced skin hydration, were relieved by the dietary administration of AM in Biostir-AD-treated NC/Nga mice. In addition, filaggrin expression increased significantly in AM-treated groups. These results suggest that AM could be a useful candidate for AD treatment.

scholarly journals Anti-Inflammatory Effects of Lasia spinosa Leaf Extract in Lipopolysaccharide-Induced RAW 264.7 Macrophages

2020 ◽  
Vol 21 (10) ◽  
pp. 3439 ◽  
Author(s):  
Thanh Q. C. Nguyen ◽  
Tran Duy Binh ◽  
Tuan L. A. Pham ◽  
Yen D. H. Nguyen ◽  
Dai Thi Xuan Trang ◽  
...  
Keyword(s):  
Leaf Extract ◽  
Inflammatory Diseases ◽  
Raw 264.7 Cells ◽  
Inflammatory Factors ◽  
Heme Oxygenase 1 ◽  
Raw 264.7 ◽  
Related Factor ◽  
Nuclear Factor ◽  
Raw 264.7 Macrophages ◽  
Anti Inflammatory

Lasia spinosa (L.) Thwaites was used as a traditional medicine to treat many inflammatory diseases for centuries. However, its effects on the inflammatory response are not yet characterized. In this study, we investigated the anti-inflammatory activities of L. spinosa leaf extract in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. We found that ethanol extracts of L. spinosa leaves showed anti-oxidant activity due to the presence of high levels of polyphenolic compounds. Treatment with the leaf extract significantly repressed the production of inflammatory mediators such as nitric oxide and reactive oxygen species and the expression of pro-inflammatory cytokines in the LPS-stimulated RAW 264.7 cells. Moreover, L. spinosa leaf extract treatment prevented activation of the nuclear factor-kappa B pathway by inhibiting nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) degradation. Furthermore, the mitogen-activated kinase and phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) pathways were suppressed upon treatment with the leaf extract. In addition to suppressing inflammatory factors, the extract also activated the nuclear factor erythroid 2-related factor 2/heme-oxygenase-1 pathway. We propose that L. spinosa leaf extract has the potential as an effective therapeutic agent for alleviating oxidative stress and excessive inflammation.

scholarly journals Inhibitor of Hyaluronic Acid Synthesis 4-Methylumbelliferone as an Anti-Inflammatory Modulator of LPS-Mediated Astrocyte Responses

2020 ◽  
Vol 21 (21) ◽  
pp. 8203 ◽  
Author(s):  
Dmitry V. Chistyakov ◽  
Arina I. Nikolskaya ◽  
Sergei V. Goriainov ◽  
Alina A. Astakhova ◽  
Marina G. Sergeeva
Keyword(s):  
Hyaluronic Acid ◽  
Inflammatory Responses ◽  
Specific Inhibitor ◽  
Acid Synthesis ◽  
Interleukin 10 ◽  
Ultra Performance Liquid Chromatography ◽  
Necrosis Factor Alpha ◽  
Inflammatory Stimuli ◽  
Anti Inflammatory ◽  
Ha Synthase

Astrocytes are glial cells that play an important role in neuroinflammation. Astrocytes respond to many pro-inflammatory stimuli, including lipopolysaccharide (LPS), an agonist of Toll-like receptor 4 (TLR4). Regulatory specificities of inflammatory signaling pathways are still largely unknown due to the ectodermal origin of astrocytes. Recently, we have shown that hyaluronic acid (HA) may form part of astrocyte inflammatory responses. Therefore, we tested 4-methylumbelliferone (4-MU), a specific inhibitor of HA synthesis, as a possible regulator of LPS-mediated responses. Rat primary astrocytes were treated with LPS with and without 4-MU and gene expression levels of inflammatory (interleukins 1β, (IL-1β), 6, (IL-6), tumor necrosis factor alpha TNFα,) and resolution interleukin 10 (IL-10) markers were evaluated via real-time PCR and western blot. The release of cytokines and HA was determined by ELISA. Oxylipin profiles were measured by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis. Our data show that 4-MU (i) has anti-inflammatory effects in the course of TLR4 activation, decreasing the cytokines level TNFα, IL-6 and IL-1β and increasing IL-10, (ii) downregulates prostaglandin synthesis but not via cyclooxygenases COX-1 and COX-2 pathways, (iii) modulates HA synthesis and decreases LPS-induced HA synthase mRNA expression (HAS-1, HAS-2) but does not have an influence on HAS-3, HYAL1 and HYAL2 mRNAs; (iv) the effects of 4-MU are predominantly revealed via JNK but not p38, ERK mitogen-activated protein kinases (MAPKs) or nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) pathways. For the first time, it is shown that 4-MU possesses the useful potential to regulate an inflammatory astrocyte response.

scholarly journals Octominin Inhibits LPS-Induced Chemokine and Pro-inflammatory Cytokine Secretion from RAW 264.7 Macrophages via Blocking TLRs/NF-κB Signal Transduction

Biomolecules ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 511 ◽  
Author(s):  
K. K. Asanka Sanjeewa ◽  
D. P. Nagahawatta ◽  
Hye-Won Yang ◽  
Jae Young Oh ◽  
Thilina U. Jayawardena ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Inflammatory Cytokine ◽  
Inflammatory Responses ◽  
Signal Transduction Pathway ◽  
Raw 264.7 Cells ◽  
Raw 264.7 ◽  
Potential Candidate ◽  
Transduction Pathway ◽  
Raw 264.7 Macrophages ◽  
In Silico Docking

Inflammation is a well-organized innate immune response that plays an important role during the pathogen attacks and mechanical injuries. The Toll-like receptors (TLR)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a major signal transduction pathway observed in RAW 264.7 macrophages during the inflammatory responses. Here, we investigated the anti-inflammatory effects of Octominin; a bio-active peptide developed from Octopus minor in RAW 264.7 macrophages in vitro. Octominin was found to inhibit lipopolysaccharides (LPS)-stimulated transcriptional activation of NF-κB in RAW 264.7 cells and dose-dependently decreased the mRNA expression levels of TLR4. Specifically, in silico docking results demonstrated that Octominin has a potential to inhibit TLR4 mediated inflammatory responses via blocking formation of TLR4/MD-2/LPS complex. We also demonstrated that Octominin could significantly inhibit LPS-induced secretion of pro-inflammatory cytokine (interleukin-β; IL-1β, IL-6, and tumor necrosis factor-α) and chemokines (CCL3, CCL4, CCL5, and CXCL10) from RAW 264.7 cells. Additionally, Octominin repressed the LPS-induced pro-inflammatory mediators including nitric oxide (NO), prostaglandin E2, inducible NO synthase, and cyclooxygenase 2 in macrophages. These results suggest that Octominin is a potential inhibitor of TLRs/NF-κB signal transduction pathway and is a potential candidate for the treatment of inflammatory diseases.

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