scholarly journals 99mTc-labeled TSPO ligand CB86 targeting macrophages for rheumatoid arthritis SPECT imaging and preliminary evaluation of anti-inflammatory effect

2019 ◽  
Author(s):  
Peng Liu ◽  
Rongshui Yang ◽  
Fu Su ◽  
Zhenyu Hou ◽  
Wentao Dong ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Radiochemical Purity ◽  
Spect Imaging ◽  
Translocator Protein ◽  
Raw264.7 Cells ◽  
Macrophage Cells ◽  
Translocator Protein 18 Kda ◽  
Anti Inflammatory ◽  
Raw264.7 Macrophage

Abstract Background and Objective TSPO (translocator protein, 18 kDa) is up-regulated in activated macrophages, and serves as an attractive target for macrophages molecular imaging. Previous studies showed that TSPO radiotracer can visualize arthritis via positron emission tomography (PET). Compared with PET, single photon emission computed tomography (SPECT) has several advantages, such as lower cost and commercial availability. The aim of the present study is to develop the 99mTc-labeled TSPO ligand CB86 as a novel SPECT probe for imaging of rheumatoid arthritis and preliminary evaluating the effectiveness of steroid anti-inflammatory therapy. Methods A novel TSPO ligand CB86 was linked to DTPAA and then labeled with 99mTc to obtain 99mTc-DTPA-CB86. The labeling efficiency, radiochemical purity, and stability were determined in vitro. In vitro cellular uptake, efflux and binding affinity of 99mTc-DTPA-CB86 to TSPO were performed on RAW264.7 macrophage cells. The distribution and SPECT studies were conducted on Freund’s Adjuvant-Induced Left Arthritis in rats after the injection of 99mTc-DTPA-CB86 with or without co-injection of unlabeled DTPA-CB86. Result The radiosynthesis of 99mTc-DTPA-CB86 was completed successfully with the labeling yields and radiochemical purity of 95.86 ± 2.45 % and 97.45 ± 0.69 %, respectively. 99mTc-DTPA-CB86 displayed good stability, which the radiochemical purity was more than > 90%, in the saline or mouse serum at 4 h. It also exhibited high specific TSPO binging in RAW264.7 macrophage cells in vitro. The highest uptake ratio was (36.45 ± 2.18) % at 3 h after incubation, and decreased significantly after adding excessive unlabeled DTPA-CB86. 99mTc-DTPA-CB86 bound to TSPO with low nanomolar affinity (IC50 =0.49 nM) in RAW264.7 cells. The cell efflux study showed that 99mTc-DTPA-CB86 has good cell retention by RAW264.7 cells, with only about 13.99 % (decreased from (33.31 ± 2.34) % to (19.32 ± 2.01) % of total input radioactivity) of 99mTc-DTPA-CB86 efflux observed during 4.5 h to 8 h incubation. Biodistribution studies showed the left inflammatory ankle uptake was 2.35±0.10 %ID/g, and the inflammatory ankle to muscle ratio was 3.01 ± 0.09 at 180 min after injection. Small animal SPET imaging studies revealed that 99mTc-DTPA-CB86 could clearly identify left inflammatory ankle with good contrast at 30-180 min after injection. Uptake of 99mTc-DTPA-CB86 in the inflammatory ankles could be largely blocked by an excess of unlabled DTPA-CB86. Furthermore, 99mTc-DTPA-CB86 accumulation in the left inflammatory ankles significantly decreased in RA rats treated with dexamethasone. Conclusion 99mTc-DTPA-CB86 can be readily synthesized, clearly visualized arthritis with low background and monitor therapy response of anti-inflammatory therapy, suggesting its potential as a novel promising molecular probe targeting TSPO for arthritic SPECT imaging. Key words TSPO (translocator protein, 18 kDa); Technetium radioisotope; SPECT imaging; Rheumatoid arthritis; Macrophages; Glucocorticoids.

scholarly journals Pentoxifylline Can Reduce the Inflammation Caused by LPS after Inhibiting Autophagy in RAW264.7 Macrophage Cells

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Danping Ruan ◽  
Sinan Deng ◽  
Zhonglong Liu ◽  
Jie He
Keyword(s):  
Phosphodiesterase Inhibitor ◽  
Inflammatory Factors ◽  
Signal Pathways ◽  
Ampk Signaling ◽  
Macrophage Cells ◽  
Raw264.7 Macrophages ◽  
Anti Inflammatory ◽  
Raw264.7 Macrophage ◽  
In Cells

Pentoxifylline (PTX), as a methylxanthine derivative and nonspecific phosphodiesterase inhibitor, has the characteristics of anti-inflammatory and partial inflammatory process inhibition. However, the regulatory effect of PTX on inflammatory cytokines is unclear. Autophagy can regulate the activation of inflammasomes and then inhibit inflammation as previously described. Our study attempts to explore the relationship between autophagy and PTX-mediated regulation of inflammasome suppression. Macrophage-like RAW264.7 cells were studied as the in vitro macrophage model. We investigated the anti-inflammatory effect caused by PTX with time and dose response against the LPS-induced inflammatory factors (TNF-α, IL-1β). Western blot detected the levels of autophagy-related proteins Beclin-1 and LC3, as well as the signal pathways of AMPK and p-AMPK. Fluorescence microscope and transmission electron microscope were used to observe the autophagy bodies in cells influenced by PTX. The autophagy in cells inhibited by PTX exhibited dose- and time-dependent effects, and PTX alleviated LPS-induced inflammation caused by retarded autophagy. Furthermore, in RAW264.7 macrophage cells, our data indicated that AMPK signaling perhaps functioned importantly in repressed autophagy. In addition, in RAW264.7 macrophages, our data suggested that AMPK signaling might play an important role in inhibiting autophagy during the process of PTX ameliorating LPS-mediated inflammation.

scholarly journals New 1,4-Dienonesteroids from the Octocoral Dendronephthya sp.

Marine Drugs ◽  
2019 ◽  
Vol 17 (9) ◽  
pp. 530 ◽  
Author(s):  
Thanh-Hao Huynh ◽  
Pei-Chin Chen ◽  
San-Nan Yang ◽  
Feng-Yu Lin ◽  
Tung-Pin Su ◽  
...  
Keyword(s):  
Inflammatory Activity ◽  
Spectroscopic Methods ◽  
Inos Protein ◽  
Macrophage Cells ◽  
Anti Inflammatory ◽  
Raw264.7 Macrophage

Two new steroids, dendronesterones D (1) and E (2), featuring with 1,4-dienone moiety, along with three known steroids, methyl 3-oxochola-4,22-diene-24-oate (3), 5α,8α-epidioxy-24(S)- methylcholesta-6,22-dien-3β-ol (4), and 5α,8α-epidioxy-24(S)-methylcholesta-6,9(11),22-trien-3β-ol (5), were isolated from an octocoral Dendronephthya sp. The structures of steroids 1 and 2 were elucidated by using spectroscopic methods and steroid 1 was found to exhibit significant in vitro anti-inflammatory activity in lipopolysaccharides (LPS)-induced RAW264.7 macrophage cells by inhibiting the expression of the iNOS protein.

scholarly journals In vitro Anti-inflammatory Evaluation of 6-Thioguanine and 6-Hydroxy-2-Mercaptopurine as A Potential Treatment for Rheumatoid Arthritis

2021 ◽  
Vol 13 (4) ◽  
pp. 383-95
Author(s):  
Nurul Syuhada Nordin ◽  
Yong Yoke Keong ◽  
Siti Farah Md Tohid ◽  
Zetty Nadia Md Zain ◽  
Muhammad Nazrul Hakim
Keyword(s):  
Rheumatoid Arthritis ◽  
Raw264.7 Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Potential Treatment ◽  
Inflammatory Reactions ◽  
Tnf Α ◽  
Ic50 Value ◽  
Anti Inflammatory ◽  
Cytotoxicity Assessment

BACKGROUND: Eventhough 6-Mercaptopurine act as a major drug for rheumatoid arthritis (RA) treatment, however, its toxicity become a limitation. Therefore, this current study investigated whether 6-hydroxy-2-mercaptopurine (6H2MP) and 6-thioguanine (6TG) compounds are purine nucleoside analogues as a potential treatment of RA. The objective was to evaluate the therapeutics effects, especially the anti-inflammatory potential of 6H2MP and 6TG in the lipopolysaccharides (LPS)-induced RAW264.7 cells and phorbol myristate acetate (PMA)-induced HIG-82 cells.METHODS: Macrophage cells (RAW264.7) and rabbit synoviocytes (HIG-82) cells were induced using LPS and PMA to evaluate the anti-inflammatory potential of 6H2MP and 6TG. The cytotoxicity assessment was done by using MTT assay, while enzyme-linked immunosorbent assay (ELISA) was used to determine the anti‑inflammatory potential, including tumour necrosis factor (TNF-α), interleukins (IL-1β, and IL-6).RESULTS: Upon LPS-induced, RAW 264.7 macrophages demonstrated that 6H2MP and 6TG could suppress the production of nitric oxide (NO) in vitro. The half-maximal inhibitory concentration (IC50) value of 6TG and 6H2MP were 10.73 and 13.31, respectively. Further studied in PMA-induced HIG-82 synovial fibroblast cells showed that 6H2MP and 6TG also suppressed the release of NO, Prostaglandin E2 (PGE2), and inflammatory cytokines such as TNF-α, IL-1β and IL-6. The 6TG is more effective to reduce inflammatory reactions compared to 6H2MP, by the lower dose needed compared to 6H2MP in all experiments except in PGE2.CONCLUSION: The inhibition of inflammatory mediators is an important mechanism by which 6TG and 6H2MP may alleviate pain and articular inflammation. These results indicated that 6H2MP and 6TG are effective candidates for ameliorating inflammatory-associated complications.KEYWORDS: anti-inflammatory, HIG-82 cells, RAW264.7 cells, 6-Thioguanine, 6-Hydroxy-2-Mercaptopurine

scholarly journals Anti-Inflammatory and Antiosteoclastogenic Activities of Parthenolide on Human Periodontal Ligament CellsIn Vitro

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Xufang Zhang ◽  
Chen Fan ◽  
Yin Xiao ◽  
Xueli Mao
Keyword(s):  
Periodontal Ligament ◽  
Tooth Loss ◽  
Nuclear Factor Kappa B ◽  
Raw264.7 Cells ◽  
Erk Activation ◽  
Tanacetum Parthenium ◽  
Coculture Model ◽  
Anti Inflammatory ◽  
Potential Use

Periodontitis is an inflammatory disease that causes osteolysis and tooth loss. It is known that the nuclear factor kappa B (NF-κB) signalling pathway plays a key role in the progression of inflammation and osteoclastogenesis in periodontitis. Parthenolide (PTL), a sesquiterpene lactone extracted from the shoots ofTanacetum parthenium, has been shown to possess anti-inflammatory properties in various diseases. In the study reported herein, we investigated the effects of PTL on the inflammatory and osteoclastogenic response of human periodontal ligament-derived cells (hPDLCs) and revealed the signalling pathways in this process. Our results showed that PTL decreased NF-κB activation, I-κB degradation, and ERK activation in hPDLCs. PTL significantly reduced the expression of inflammatory (IL-1β, IL-6, and TNF-α) and osteoclastogenic (RANKL, OPG, and M-CSF) genes in LPS-stimulated hPDLCs. In addition, PTL attenuated hPDLC-induced osteoclastogenic differentiation of macrophages (RAW264.7 cells), as well as reducing gene expression of osteoclast-related markers in RAW264.7 cells in an hPDLC-macrophage coculture model. Taken together, these results demonstrate the anti-inflammatory and antiosteoclastogenic activities of PTL in hPDLCsin vitro. These data offer fundamental evidence supporting the potential use of PTL in periodontitis treatment.

Ulinastatin Activated The ERK5/Mer Signaling Pathway To Promote Macrophage Efferocytosis And Ameliorate Lung Inflammation

2021 ◽  
Author(s):  
Jinju Li ◽  
Rongge Shao ◽  
Qiuwen Xie ◽  
XueKe Du
Keyword(s):  
Lung Injury ◽  
Signaling Pathway ◽  
Lung Inflammation ◽  
Raw264.7 Cells ◽  
Inflammatory Factors ◽  
Inflammatory Factor ◽  
Dependent Manner ◽  
Anti Inflammatory

Abstract Purpose:Ulinastatin (UTI) is an endogenous protease inhibitor with potent anti-inflammatory, antioxidant and organ protective effects. The inhibitor has been reported to ameliorate inflammatory lung injury but precise mechanisms remain unclear. Methods: An in vivo model of lung injury has been constructed by intratracheal infusion of lipopolysaccharide (LPS). The number of neutrophils and the phagocytosis of apoptotic neutrophils were observed by Diff- Quick method. Lung injury was observed by HE staining .BALF cells were counted by hemocytometer and concentrations of protein plus inflammatory factors were measured with a BCA test kit. During in vitro experiments, RAW264.7 cells were pretreated with UTI (1000 and 5000U/ mL), stained with CellTrackerTM Green B0DIPYTM and HL60 cells added with UV-induced apoptosis and PKH26 Red staining. The expression of ERK5\Mer related proteins was detected by western blot and immunofluorescence.Results: An in vivo model of lung injury has been constructed by intratracheal infusion of lipopolysaccharide (LPS). UTI treatment enhanced the phagocytotic effect of mouse alveolar macrophages on neutrophils, alleviated lung lesions, decreased the pro-inflammatory factor and total protein content of BALF and increased levels of anti-inflammatory factors. in vitro experiments ，UTI enhanced the phagocytosis of apoptotic bodies by RAW264.7 cells in a dose-dependent manner. Increased expression levels of ERK5 and Mer by UTI were shown by Western blotting and immunofluorescence.Conclusions: UTI mediated the activation of the ERK5/Mer signaling pathway, enhanced phagocytosis of neutrophils by macrophages and improved lung inflammation. The current study indicates potential new clinical approaches for accelerating the recovery from lung inflammation.

scholarly journals Anti-Inflammatory Effects of Formononetin 7-O-phosphate, a Novel Biorenovation Product, on LPS-Stimulated RAW 264.7 Macrophage Cells

Molecules ◽  
2019 ◽  
Vol 24 (21) ◽  
pp. 3910 ◽  
Author(s):  
Min-Seon Kim ◽  
Jin-Soo Park ◽  
You Chul Chung ◽  
Sungchan Jang ◽  
Chang-Gu Hyun ◽  
...  
Keyword(s):  
Biological Properties ◽  
In Vitro Assays ◽  
Raw 264.7 Cells ◽  
No Production ◽  
Raw 264.7 ◽  
Dependent Manner ◽  
Macrophage Cells ◽  
Anti Inflammatory ◽  
Reduced Toxicity

Biorenovation is a microbial enzyme-catalyzed structural modification of organic compounds with the potential benefits of reduced toxicity and improved biological properties relative to their precursor compounds. In this study, we synthesized a novel compound verified as formononetin 7-O-phosphate (FMP) from formononetin (FM) using microbial biotransformation. We further compared the anti-inflammatory properties of FMP to FM in lipopolysaccharide (LPS)-treated RAW264.7 macrophage cells. We observed that cell viabilities and inhibitory effects on LPS-induced nitric oxide (NO) production were greater in FMP-treated RAW 264.7 cells than in their FM-treated counterparts. In addition, FMP treatment suppressed the production of proinflammatory cytokines such as prostaglandin-E2 (PGE2), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in a dose-dependent manner and concomitantly decreased the mRNA expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2). We also found that FMP exerted its anti-inflammatory effects through the downregulation of the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and nuclear factor kappa B (NF-κB) signaling pathways. In conclusion, we generated a novel anti-inflammatory compound using biorenovation and demonstrated its efficacy in cell-based in vitro assays.

scholarly journals Antioxidant and Anti-Inflammatory Activities of Six Flavonoids from Smilax glabra Roxb

Molecules ◽  
2020 ◽  
Vol 25 (22) ◽  
pp. 5295
Author(s):  
Xinyu Zhao ◽  
Ruyi Chen ◽  
Yueyue Shi ◽  
Xiaoxi Zhang ◽  
Chongmei Tian ◽  
...  
Keyword(s):  
Antioxidant Activities ◽  
Radical Scavenging Activity ◽  
Radical Scavenging ◽  
Raw264.7 Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Preparative Hplc ◽  
Antioxidant Power ◽  
Ferric Reducing Antioxidant Power ◽  
Anti Inflammatory

This study aimed to isolate, prepare and identify the main flavonoids from a standardized Smilax glabra flavonoids extract (SGF) using preparative HPLC, MS, 1H NMR and 13C NMR, determine the contents of these flavonoids using UPLC, then compare their pharmacological activities in vitro. We obtained six flavonoids from SGF: astilbin (18.10%), neoastilbin (11.04%), isoastilbin (5.03%), neoisoastilbin (4.09%), engeletin (2.58%) and (−)-epicatechin (1.77%). The antioxidant activity of six flavonoids were evaluated by determining the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and 2,2′-Azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS+) radical scavenging activity and ferric reducing antioxidant power (FRAP). In addition, the anti-inflammatory activity of six flavonoids were evaluated by determining the production of cytokines (IL-1β, IL-6), nitric oxide (NO) using enzyme linked immunosorbent assay and the NF-κB p65 expression using Western blotting in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. The results showed that (−)-epicatechin, astilbin, neoastilbin, isoastilbin and neoisoastilbin had strong antioxidant activities, not only in DPPH and ABTS+ radicals scavenging capacities, but in FRAP system. Furthermore, all the six flavonoids could significantly inhibit the secretion of IL-1β, IL-6, NO (p < 0.01) and the protein expression of NF-κB p-p65 (p < 0.01) in LPS-stimulated RAW264.7 cells. This study preliminarily verified the antioxidant and anti-inflammatory activities of six flavonoids in S. glabra.

Anti-Inflammatory Effects of Canavalia gladiata in Macrophage Cells and DSS-Induced Colitis Mouse Model

2019 ◽  
Vol 47 (07) ◽  
pp. 1571-1588
Author(s):  
Hwa-Jeong Lee ◽  
Jung Up Park ◽  
Rui Hong Guo ◽  
Bok Yun Kang ◽  
In-Kyu Park ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nuclear Translocation ◽  
Interleukin 1 ◽  
Raw264.7 Cells ◽  
Mice Model ◽  
Macrophage Cells ◽  
Canavalia Gladiata ◽  
Cox 2 ◽  
Anti Inflammatory ◽  
Peritoneal Macrophage Cells

Canavalia gladiata, known as sword bean, has been used as a Chinese traditional medicine for anti-inflammatory effects. However, the action mechanisms of sword bean have not yet been clearly defined. In the present study, the whole parts of a ripened sword bean (RSB) and the green sword bean (GSB) containing bean pod were extracted with ethanol by reflux extraction. The two crude extracts (RSBE and GSBE) from RSB and GSB were validated by a liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis of gallic acid as a reference chemical. The anti-inflammatory effects of two sword bean extracts were extensively investigated using LPS-stimulated macrophage cells. First, RSBE and GSBE significantly inhibited the production of pro-inflammatory mediators, such as tumor necrosis factor-[Formula: see text] (TNF-[Formula: see text]), interleukin-6 (IL-6), prostaglandinE2 (PGE2), and nitric oxide (NO) in LPS-induced RAW264.7 cells. RSBE and GSBE showed no cytotoxicity to RAW264.7 cells and mouse peritoneal macrophage cells. In addition, the overexpression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) induced by LPS in RAW264.7 cells was significantly decreased by RSBE and GSBE. Western blotting and immunostaining analysis showed that RSBE and GSBE inhibited the nuclear translocation of NF-[Formula: see text]B subunits, which correlated with the inhibitory effects on inhibitor kappa B (I[Formula: see text]B) degradation. In dextran sulfated sodium (DSS)-induced colitis mice model, RSBE restored body weight, colon length, and the levels of pro-inflammatory cytokines, such as TNF-[Formula: see text], IL-6, interleukin-1[Formula: see text] (IL-1[Formula: see text]), and interferon-[Formula: see text] (IFN-[Formula: see text]). In addition, RSBE significantly suppressed the expression of COX-2, iNOS, and NF-[Formula: see text]B.

scholarly journals Dual Effect of Soloxolone Methyl on LPS-Induced Inflammation In Vitro and In Vivo

2020 ◽  
Vol 21 (21) ◽  
pp. 7876
Author(s):  
Andrey V. Markov ◽  
Aleksandra V. Sen’kova ◽  
Valeriya O. Babich ◽  
Kirill V. Odarenko ◽  
Vadim A. Talyshev ◽  
...  
Keyword(s):  
Acute Inflammation ◽  
Nuclear Factor Κb ◽  
Raw264.7 Cells ◽  
Migration Activity ◽  
Akt Phosphorylation ◽  
Raw264.7 Macrophages ◽  
Anti Inflammatory ◽  
And Migration

Plant-extracted triterpenoids belong to a class of bioactive compounds with pleotropic functions, including antioxidant, anti-cancer, and anti-inflammatory effects. In this work, we investigated the anti-inflammatory and anti-oxidative activities of a semisynthetic derivative of 18βH-glycyrrhetinic acid (18βH-GA), soloxolone methyl (methyl 2-cyano-3,12-dioxo-18βH-olean-9(11),1(2)-dien-30-oate, or SM) in vitro on lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and in vivo in models of acute inflammation: LPS-induced endotoxemia and carrageenan-induced peritonitis. SM used at non-cytotoxic concentrations was found to attenuate the production of reactive oxygen species and nitric oxide (II) and increase the level of reduced glutathione production by LPS-stimulated RAW264.7 cells. Moreover, SM strongly suppressed the phagocytic and migration activity of activated macrophages. These effects were found to be associated with the stimulation of heme oxigenase-1 (HO-1) expression, as well as with the inhibition of nuclear factor-κB (NF-κB) and Akt phosphorylation. Surprisingly, it was found that SM significantly enhanced LPS-induced expression of the pro-inflammatory cytokines interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) in RAW264.7 cells via activation of the c-Jun/Toll-like receptor 4 (TLR4) signaling axis. In vivo pre-exposure treatment with SM effectively inhibited the development of carrageenan-induced acute inflammation in the peritoneal cavity, but it did not improve LPS-induced inflammation in the endotoxemia model.

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